Methods of and compositions for inhibiting the proliferation of mammalian cells

ABSTRACT

A method of preventing, inhibiting and/or reversing cell motility, actin filament assembly or disassembly, proliferation, colonization, differentiation, accumulation and/or development of abnormal cells in a subject is disclosed. The method is effected by administering to the subject a therapeutically effective amount of a ribonuclease of the T2 family having actin binding activity.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/890,762 filed on Sep. 27, 2010, which is a continuation of U.S.patent application Ser. No. 10/952,495 filed on Sep. 29, 2004, now U.S.Pat. No. 7,811,981, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/069,454 filed Feb. 26, 2002, now U.S. Pat. No.7,101,839, which is a National Phase of PCT Patent Application No.PCT/IL00/00514 filed on Aug. 29, 2000, which is a continuation-in-partof U.S. patent application Ser. No. 09/385,411 filed Aug. 30, 1999, nowabandoned. The contents of the above applications are incorporatedherein by reference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the use of a ribonuclease of the T2family or a polynucleotide encoding same for preventing, inhibitingand/or reversing proliferation, colonization, differentiation and/ordevelopment of abnormally proliferating cells in a subject. The presentinvention further relates to pharmaceutical compositions containing, asan active ingredient, a ribonuclease of the T2 family or apolynucleotide encoding same for treating proliferative diseases ordisorders in general and cancer in particular.

There is an ongoing interest, both within the medical community andamong the general population, in the development of novel therapeuticagents for the treatment of cell proliferative diseases and disorderssuch as cancer.

Agents that display anti-proliferative, anti-colonization,anti-differentiation and/or anti-development properties againstmammalian cells can potentially be used as anti-cancer drugs. As such,these agents are widely sought for from both natural as well assynthetic sources.

RIBASES are ribonucleases (RNases) which display a biological activitywhich is distinct from their ability to degrade RNA. RIBASES and theirstructural homologous are known to effect a large number of cellularreactions (Rybak, M. et al., 1991, J. Biol. Chem. 266:21202-21207;Schein, C. H. 1997 Nature Biotechnol. 15:529-536). EDN and ECP, twomajor proteins found in the secretory granules of cytotoxic eosinophyles(members of RNase A family) are thought to participate in the immuneresponse. In self-incompatible plants stylar S-RNases (members of RNaseT2 family), arrest pollen tube growth and thus prevent fertilization.RC-RNase, produced from Bullfrog oocytes, inhibits, in vitro, the growthof tumor cells such as the P388, and L1210 leukemia cell lines and iseffective for in vivo killing of sarcoma 180, Erlich, and Mep II ascitescells (Chang, C-F. et al 1988, J. Mol. Biol 283:231-244). Some RNasesdisplay limited ribonuclease activity, an example of which includesangiogenins that stimulate blood vessels formation (Fett, J. W. 1985,Biochemistry 24:5480-5486).

Living organisms use extracellular RNases for defense against pathogensand tumor cells. For example, ECP is secreted in response to parasiteattack (Newton, D L. 1992, J. Biol. Chem. 267:19572-19578) and displaysantibacterial and antiviral activity. This activity is also displayed byZinc-α₂-glycoprotein (Znα₂gp), an RNase present in most human bodyfluids including blood, seminal plasma, breast milk, synovial fluid,saliva, urine and sweat (Lei G, et al., 1998, Arch Biochem Biophys. July15; 355(2):160-4).

The specific mechanism by which extracellular RNases function incellular reactions is unknown.

The main barrier to the cytotoxic activity of some RNase is the cellmembrane. ECP was found to form channels in both artificial and cellularmembranes. Presumably, ECP released from the granule membrane along withEDN (eosinophylic RNase, which is responsible for cerebellar Purkinjiecell destruction) transfers EDN into the intercellular space. Theentrance of the fungal toxin α-sarcin (a member of the RNase A family)into target cells depends upon viral infection which permeabilizes thecellular membrane (Rybak, M. et al., 1991, J. Biol. Chem.266:21202-21207). It is also possible that RNases enter the cell viaendocytosis. When the Golgi-disrupting drugs retinoic acid or monensinwere used to artificially deliver BS-RNase into the cells, cytotoxicityincreased dramatically (Wu Y, et al., 1995, J Biol. Chem. 21;270(29):17476-81).

Cytotoxicity of RNases can be used for therapeutic purposes. Human RNaseL is activated by interferon and inhibits viral growth. Expression ofthe gene for human RNase L together with that for a 2′5′-A synthetase intobacco plants is sufficient to protect plants from cucumber mosaicvirus and to prevent replication of potato virus Y. Humanimmunodeficiency virus-1 (HIV-1) induces blockade in the RNase L toantiviral pathways (Schein, C. H. 1997 Nature Biotechnol. 15:529-536.).RNases can be fused with specific membranal protein antibodies to createimmunotoxins. For example, fusion of RNase A with antibodies to thetransferrin receptor or to the T cell antigen CD5 lead to inhibition ofprotein synthesis in tumor cells carrying a specific receptor for eachof the above toxins (Rybak, M. et al., 1991, J. Biol. Chem.266:21202-21207; Newton D L, et al., 1998, Biochemistry 14;37(15):5173-83). Since RNases are less toxic to animals, they may havefewer undesirable side effect than the currently used immunotoxins.

The cytotoxicity of cytotoxic ribonucleases appears to be inverselyrelated to the strength of the interaction between a ribonucleaseinhibitor (RI) and the RNase. Ribonuclease inhibitor (RI) is a naturallyoccurring molecule found within vertebrate cells which serves to protectthese cells from the potentially lethal effects of ribonucleases. Theribonuclease inhibitor is a 50 kDa cytosolic protein that binds toRNases with varying affinity. For example, RI binds to members of thebovine pancreatic ribonuclease A (RNase A) superfamily of ribonucleaseswith inhibition constants that span ten orders of magnitude, withK_(i)'s ranging from 10⁻⁶ to 10⁻¹⁶ M.

A-RNases

ONCONASE, like RNase A and BS-RNase, is a member of the RNase Asuperfamily. Members of the RNase A superfamily share about 30% identityin amino acid sequences. The majority of non-conserved residues arelocated in surface loops, and appear to play a significant role in thededicated biological activity of each RNase. ONCONASE was isolated fromNorthern Leopard frog (Rana pipiens) oocytes and early embryos. It hasanti-tumor effect on a variety of solid tumors, both in situ and in vivo(Mikulski S. M., et al., 1990 J. Natl. Cancer 17; 82(2):151-3). ONCONASEhas also been found to specifically inhibit HIV-1 replication ininfected H9 leukemia cells at non-cytotoxic concentration (Youle R. J.,et al., 1994, Proc. Natl. Acad. Sci. 21; 91(13):6012-6).

Although the RNase activity of ONCONASE is relatively low, it isaccepted that the enzymatic and cytotoxic activities thereof areassociated to some degree. It is believed that the tertiary structure ofA-RNases differentiate between cytotoxic and non-cytotoxic types. Forexample, differences between the tertiary structure of ONCONASE andRNase A are believed to be responsible for the increased cytotoxicityobserved for ONCONASE. ONCONASE, unlike RNase A, contains a blockedN-terminal Glu1 residue (pyroglutamate) which is essential for bothenzymatic and cytotoxic activities. This unique structure enablesONCONASE to permeate into target cells (Boix E., et al., 1996, J. Mol.Biol. 19:257(5):992-1007). In addition, in ONCONASE the Lys9 residuereplaces the Gln11 residue of RNase A, which is believed to effect thestructure of the active site. Furthermore, differences in the amino acidsequence of the primary structure between ONCONASE and RNase A causetopological changes at the periphery of the active site which effect thespecificity thereof (Mosimann S. C., et al., 1992, Proteins14(3):392-400).

The differences in toxicity between A-RNases are also attributed totheir ability to bind RI. Bovine seminal ribonuclease (BS-RNase) is 80%identical in its amino acid sequence to RNase A, but unlike othermembers of the RNase A superfamily, BS-RNase exists in a dimeric form.It has been shown that the quaternary structure of BS-RNase preventsbinding by RI, thereby allowing the enzyme to retain its ribonucleolyticactivity in the presence of RI (Kim et al., 1995, J. Biol. Chem. 270 No.52:31097-31102). ONCONASE, which shares a high degree of homology withRNase A, is resistant to binding by RI. The RI-ONCONASE complex has aK_(d) at least one hundred million times less than that of the RI-RNaseA complex. The lower binding affinity of ONCONASE for RI preventseffective inhibition of the ribonucleolytic activity and could explainwhy ONCONASE is cytotoxic at low concentrations while RNase A is not.

It is suggested that binding to cell surface receptor is the first stepin ONCONASE cytotoxicity. Nothing is known about the nature of ONCONASEreceptors on mammalian cell surfaces. ONCONASE may bind to cell surfacecarbohydrates as in the case of ricin, or it may bind to receptorsoriginally developed for physiologically imported molecules likepolypeptide hormones (Wu Y, et al., 1993, J. Biol. Chem. 15;268(14):10686-93). In mice, ONCONASE was eliminated from the kidneys ina rate 50-100-fold slower than did RNase A. The slower elimination rateof ONCONASE is explained as a result of its higher ability to bind tothe tubular cells and/or by its resistance to proteolytic degradation.The strong retention of ONCONASE in the kidneys might have clinicalimplications (Vasandani V. M., et al., 1996, Cancer Res. 15;56(18):4180-6). ONCONASE may also bind to Purkinjie cells EDN receptors(Mosimann S. C., et al., 1996, J. Mol. Biol. 26; 260(4):540-52). Thespecificity of ONCONASE is also expressed in its tRNA preference. Inrabbit reticulocyte lysate and in Xenopus oocytes it was discovered thatONCONASE inhibits protein synthesis via tRNA, rather than via rRNA ormRNA degradation. In contrast, RNase A degrades mostly rRNA and mRNA(Lin J. J., et al., 1994, Biochem. Biophys. Res. Commun 14;204(1):156-62).

Treatment of susceptible tissue cultures with ONCONASE results in theaccumulation of cells arrested in G1 phase of the cell cycle, havingvery low level of RNA contents (Mosimann S. C., et al., 1992, Proteins14(3):392-400). In glioma cells ONCONASE inhibited protein synthesiswithout a significant reduction in cell density, showing that ONCONASEis also cytotoxic to cells in addition to being cytostatic (Wu Y., etal., 1993, J. Biol. Chem. 15; 268(14):10686-93). ONCONASE, combined withchemotherapeutic agents, can overcome multidrug resistance. Treatmentwith vincristine and ONCONASE increased the mean survival time (MST) ofmice carrying vincristine resistant tumors to 66 days, compared to 44days in mice treated with vincristine alone (Schein, C. H., 1997, NatureBiotechnol. 15:529-536).

Furthermore, some chemotherapeutic agents may act in synergy withONCONASE. In tumor cell lines of human pancreatic adenocarcinoma andhuman lung carcinoma treated with a combination of ONCONASE andtamoxifen (anti-estrogen), trifluoroperazine (Stelazine, calmodulininhibitor) or lovastatin (3-hydroxyl-3-methylglutatyl coenzyme A(HMG-CoA) reductase inhibitor) a stronger growth inhibition was observedthan cells treated with ONCONASE alone (Mikulski S. M., et al., 1990,Cell Tissue Kinet. 23(3):237-46). Thus, a possibility of developingcombination therapy regiments with greater efficiency and/or lowertoxicity is clear.

Bovine seminal RNase is a unique member of RNase A family, since it isthe only RNase containing a dimmer of RNase A-like subunits linked bytwo disulfide bridges. In addition, it maintains allosteric regulationby both substrate and reaction products. The regulation occurs at thecyclic nucleotide hydrolysis phase. It has the ability to cleave bothsingle- and double-stranded RNA. BS-RNase is highly cytotoxic. Itdisplays anti-tumor effect in vitro on mouse leukemic cells, HeLa andhuman embryo lung cells, mouse neuroblastoma cells, and humanfibroblasts and mouse plasmacytoma cell lines. When administrated invivo to rats bearing solid carcinomas (thyroid follicular carcinoma andits lung metastases), BS-RNase induced a drastic reduction in tumorweight, with no detectable toxic effects on the treated animals(Laccetti, P. et al., 1992, Cancer Research 52:4582-4586). Artificiallymonomerized BS-RNase has higher ribonuclease activity but lowercytotoxicity than native dimeric BS-RNase (D'Allessio G., et al., 1991,TIBS:104-106). This, again, indicates the importance of molecularstructure for the biological activity. It seems that like ONCONASE,BS-RNase binds to recognition site(s) on the surface of the targetcells, prior to penetration into target cells.

In addition to being cytotoxic, BS-RNase is also immunorepressive.BS-RNase can block the proliferation of activated T cells, and prolongthe survival of skin grafts transplanted into allogenetic mice. Theimmunorepressive activity of SB-RNase is explained by the need toprotect sperm cells from the female immune system.

T2-RNases

In plants, self-compatibility is abundant and is effective in preventingself-fertilization. Pollen carrying a particular allele at the S locus,which controls self-incompatibility, is unable to fertilize plantscarrying the same S-allele. In many self-incompatible plants, especiallymembers of Solanaceae and Rosaceae, S-RNase, a member of the T2-RNasefamily is secreted by the female organs. S-RNase specifically recognizeself-pollen and arrest its growth in the stigma or style beforefertilization occurs (Clarke, A. E. and Newbigin, E., 1993, Ann. Rev.Genet. 27:257-279) it is believed that the arrest of pollen tube growthis a direct consequence of RNA degradation, however the mode of S-RNaseentrance into the tube cell is still obscure.

Members of RNase T2 family were first identified in fungi (Egami, F. andNakamura, K. 1969, Microbial ribonucleases. Springer-Verlag, Berlin).Since, they were found in a wide variety of organisms, ranging fromviruses to mammals. In particular, T2-RNases show much broaderdistribution than the extensively described RNase A family. However, thein vivo role of T2-RNases in mammalian cells is still not known.

In microorganisms, extracellular T2-RNases are generally accepted tocontribute to the digestion of polyribonucleotides present in the growthmedium, thereby giving rise to diffusible nutrients. They may also serveas defense agents (Egami, F. and Nakamura, K., 1969, Microbialribonucleases. Springer-Verlag, Berlin).

In plants, T2-RNases play a role in the pollination process, byselectively limiting the elongation of pollen tubes racing towards theovules (Roiz, L. and Shoseyov, O, 1995, Int. J. Plant Sci. 156:37-41,Roiz L. et al., 1995, Physiol. Plant. 94:585-590). To date, themechanism by which these RNases affect pollen tubes is unclear.

Thus, there exist few examples of cytotoxic ribonucleases which can beeffectively used as cancer treatment agents. New ribonucleases withanti-proliferation, anti-colonization, anti-differentiation and/oranti-development activities toward mammalian cells are needed to enhancethe spectrum of therapeutic agents available for treatment of humancancers, to thereby open new horizons in the field of cancer treatment.

Apoptosis and Disease

Cell death can occur through two different processes, termed necrosisand apoptosis, which can be distinguished by specific sets of functionaland morphologic characteristics. Necrosis is a traumatic cell death thatoccurs as a response to injurious agents in the extracellularsurroundings e.g. hypoxia, hyperthermia, viral invasion, exposure totoxins, or attack by pathogens. The ion and water pumps in the plasmamembrane lose their abilities to maintain concentration gradients, thecells and mitochondria swell and eventually burst, leaking cellularconstituents and leading to an inflammatory response in the surroundingtissue. In apoptosis, or programmed cell death (PCD), cells are inducedto self-destruct via an intrinsic genomic program. The cells shrink andthe mitochondria break down and release cytochrome c. The nuclear DNA isgradually degraded into monomers and multimers of about 200 bases.Eventually, the cells undergo blebbing, and break into small,membrane-wrapped fragments called apoptotic bodies which are engulfed bynearby phagocytotic cells (Rudin C M and Thompson C B. 1997. Annu RevMed. 48:267-81; Chamond R. R. et al. 1999. Alergol Immunol Clin.14:367-374). Significantly, no inflammatory response in surroundingtissues is elicited. The difference between death forms is summarized inthe following Table 1.

TABLE 1 Apoptosis vs. Necrosis Necrosis Apoptosis Etiology Acute cellinjury due to extracellular Various intracellular or extracellularstimuli stimuli Character Pathologic Physiologic or pathologicDistribution Groups of cells or patches Wildly scattered isolated cellsof tissues Energy requirement Passive process (ATP-independent) Activeprocess (ATP-dependent) Morphologic features Swelling of the cytoplasm.Shrinkage of the cytoplasm, Membrane lysis with loss of cell andExternalization of phosphatidylserine. organelles contents. Cellmembrane blebbing to form apoptotic bodies encompassing cytoplasm andorganelles. Chromatin condensation and DNA fragmentation into multimersof 200 bases. Reaction of the Inflammation Phagocytosis withoutinflammatory reaction surrounding tissue (Nikitakis N. G. et al. 2004.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 97: 476-90.)

Apoptosis plays a central role in the regulation of homeostasis, innormal, and in pathological processes. During embryogenesis apoptosis isresponsible for the disappearance of the tadpoles tail, thedifferentiation of fingers and toes, and the removal of unnecessaryneurons in the brain. It is also involved in disassembly of theendometrium at the menstruation, and in the aging process (Herndon F J.et al. 1997. Mechanism of ageing and development 94:123-134).

Apoptosis is needed to remove cells that represent a threat to theintegrity of the organism. For example, it is the mechanism by whichcytotoxic T lymphocytes (CTLs) kill virus-infected cells (Barber G N.2001. Cell Death Differ. 8:113-126.). CTLs can induce apoptosis even ineach other, so they can be eliminated after completion of theirphysiological function, preventing their becoming a liability forsurrounding tissue (Duke R C. 1992. Semin Immunol. 4:407-412).

The anterior chamber of the eye and the testes are known as “immune toprivileged” organs, as it has been found antigens do not elicit animmune response in these sites. In these sites, the cells constitutivelyexpress high levels of Fas ligand (FasL), a cytokine that binds to acell-surface receptor named Fas (also called CD95) and known as a potentdeath activator. FasL is toxic to T cells, and thus permits theprolonged, and sometimes permanent, survival of foreign tissue and tumorgrafts (inhibited apoptosis) (Niederkorn J Y. 2002. Crit. Rev Immunol.22:13-46; Takeuchi T. et al. 1999. J. Immunol. 162:518-522; Sugihara A,et al. 1997. Anticancer Res. 17:3861-3865). Thus, upregulation ofapoptotic processes in specific cells, for example lymphocytes, can beuseful in the prevention of graft rejection, potentially leading toreduction in the use of immunosuppressive drugs and improvement in thequality of the patient's life.

A variety of diseases have been associated with regulation of apoptosis.Among them are various neurodegenerative diseases, among themAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,retinitis pigmentosa, and epilepsy, all associated with selectiveapoptosis of the neurons. This neuronal death appears to be associatedto increase susceptibility to apoptosis in these cells.

Mature blood cells are derived from haematopoietic precursors located inthe bone marrow. Haematopoiesis, as well as maintenance of mature bloodcells are regulated by a number of trophic factors (erythropoyetin,colony stimulating factors, cytokines). The balance betweenhematopoietic cell production and elimination is regulated by apoptosis.Loss of apoptosis regulation can be associate with a variety of blooddisorders e.g. aplastic anemia, myelodyplastic syndrome, CD4⁺ T cellslymophocytopenia and G6PD deficiency.

In myocardial infarction and cerebrovascular accidents, ischaemic renaldamage and polycystic kidney the cells surrounding the ischaemic zoneare eliminated through apoptosis.

There is evidence showing that apoptosis is upregulated in a variety ofcells e.g. neurons, myocytes, lymphocytes, hepatocytes, and that agingenhances apoptosis under physiological conditions that cause homeostasisdysfunction, such as oxidative stress, glycation, and DNA damage.

Apoptosis has been described in inflammatory cells (eosinophils,neutrophils, lymphocytes, macrophages, mast cells) that participate inthe late and chronic stages of allergy (Sampson A P. 2000. Clin ExpAllergy. 30 Suppl 1:22-7; Haslett C. 1999. Am J Respir Crit. Care Med.160:S5-11). For example, apoptotic death of the eosinophils isassociated with bronchial asthma, allergic rhinitis and atopicdermatitis (Wooley K L et al. 1996. Am J Respir Crit. Care Med; 154:237-243; Boyce J A. Allergy Asthma Proc. 18: 293-300). Lymphocytesapoptosis may induced by allergens, such as Olea europaea and Loliumperenneinduce (Guerra F et al. 1999. Hum Immunol; 60: 840-847).

Diseases associated with inhibition of apoptosis include those diseasesin which an excessive accumulation of cells occurs (neoplastic diseases,autoimmune diseases). Where it was once believed that the excessiveaccumulation of cells in these diseases was due to an increased cellproliferation, it is now thought to be due to defective apoptosis.

In both solid and haematological tumors, the malignant cells show anabnormal response to apoptosis inducers (Watson A J M. 1995. Gut 37:165-167; Burch W. et al. 1992. Trends Pharmacol Sci 13:245-251). Inthese diseases cycle-regulating genes such as p53, ras, c-myc and bcl-2suffer mutations, inactivation or dysregulations associated to malignantdegeneration (Merrit A J et al. 1994. Cancer Res 54:614-617; Iwadate Yet al. 1996. Int J Cancer 69:236-240; Müllauer L et al. 1996. Hepatology23: 840-847; Newcomb E W. 1995. Leuk Lymphoma 17: 211-221). Theexpression of bcl-2 is considered to be a predictive factor for worseprognosis in prostate and colonic cancer and in neuroblastoma (ThompsonC B. 1995. Science 267: 1456-1462). It has been shown that a number ofantineoplastic therapies induce apoptosis in tumour cells (for reviewssee: Sun S Y et al. 2004. J Natl Cancer Inst. 96:662-672;Schulze-Bergkamen H and Krammer P H. 2004. Semin Oncol. 31:90-119; AbendM. 2003. Int J Radiat Biol. 79:927-941).

Defects in the apoptosis may lead to autoimmune diseases such as lupuserythematosus (Carson D A. and Rebeiro J M. 1993. Lancet. 341:1251-1254. Aringer M. et al. 1994. Arthritis Rheum. 37:1423-1430),rheumatoid arthritis (Liu H. and Pope R M. 2003. Curr Opin Pharmacol.3:317-22.) and myasthenia gravis (Masunnaga A. et al. 1994. ImmunolLett. 39: 169-172.).

There are several ways by which the pathogens interfere with apoptosis.For example adenovirus and Epstein-Barr virus (associated with severallymphoid and to epithelial malignancies) promote expression of Bcl-2oncogene (Thompson C B. 1995. Science 267:1456-1462; Marshall W L. etal. 1999. J. Virol. 73:5181-5185), cowpox encode a protease inhibitorthat inactivates caspases (Deveraux Q L, et al. 1999. J Clin Immunol.19:388-98.); chlamydia interferes with mitochondrial cytochrome crelease into the cytosol (Fan T. et al. 1998. J Exp Med. 187:487-496).

In chronic inflammatory, hyperproliferative skin diseases such aspsoriasis, an abnormally low rate of apoptosis contributes to thedevelopment of epidermal hyperplasia. It was shown that keratinocytesrespond to a variety of external and internal growth factors, includingsome proinflammatory cytokines which may suppress keratinocytesapoptosis, such as IL-15 (Ruckert R. et al. 2000. J. Immunol.165:2240-2250).

Actin and Cell Motility

Actin is ubiquitous in nature, comprising the cytoskeleton and providingmotility in all types of cells. The cellular actin cytoskeleton isorganized in a variety of spatially and temporally controlled assembliesof actin filaments. Actin filaments are polymerized from monomericG-actin in lamellipodia and filopodia at the cell periphery. These newlypolymerized actin filaments are highly dynamic and are turned overrapidly (Wang, 1985). The actin filaments found in the remainder of thecells have their origin in the lamellipodium and in small membraneruffles occurring throughout the lamella. Actin filaments are organizedinto various arrays such as stress fibers, lamellipodial networks,filopodial bundles, dorsal arcs, peripheral concave or convex bundles aswell as geodesic arrays (Small, et al. Trends in Cell Biol 2002;12:112-20). The organization of each of these assemblies is controlledand stabilized by specific sets of actin-associated proteins, conferringon them different functions. An asymmetric and polarized organization ofthe different actin arrays in cells is fundamental for cell migration,growth, division, differentiation, and defense (Hilpela et al, Mol CellBiol 2003; 14:3242-53).

Cell motility depends on the cyclic dynamics of polymerization anddepolymerization of the actin cytoskeleton. Cell motility involvesprotrusion of a cell front and subsequent retraction of the rear.Protrusion is based on the forward, cyclic growth, or polymerization ofactin filaments in lamellipodia and filopodia. Retraction, on the otherhand, is based on the interaction of preformed actin filaments withmyosin-II in contractile bundles. Microscopic studies have shown acontinuum of retrograde flow of actin behind lamellipodia, indicatingthat a proportion of filaments generated in the lamellipodium contributeto the network of actin that makes up the rest of the actincytoskeleton. Thus, actin filaments are generated in the lamellipodia,shed their associate proteins as they become incorporated into the actincytoskeleton, and then acquire other actin-associated proteins,particularly contractile proteins such as myosin-II, becomingcontractile bundles in preparation for retraction of the cellprotrusions.

Cell shape change and motility are involved in pathological events, suchas cancer metastasis, inflammatory disease, neurodegenerative diseaseand the like. Cell motility associated proteins have been identified inthe pathogenesis of a number of diseases, such as Wiskott-AldrichSyndrome (WAS protein).

A large and growing number of proteins are known to regulate andmodulate the state of the actin cytoskeleton, and some appear to havepartly overlapping functions. These include actin and integrin bindingproteins such as filamin, talin, Arp 2/3 complex, α-actinin, filamentsevering proteins and barbed end capping proteins (for review, seeBrakebusch et al, EMBO Journal, 2003; 22:2324-33). In addition, thereexist proteins of different upstream signaling pathways leading tochanges in the actin cytoskeleton and cell morphology and behavior suchas the small Ras-related GTPases, e.g., Rac, Rho, and Cdc42. In additionto these small GTPases, phosphoinositides and calcium are known toregulate actin dynamics and cell migration.

Presently, very few specific inhibitors of cell motility are available,even though a great potential exists for such drugs as a complement toexisting therapies for inflammatory disease, cancer, neurodegenerativedisease and the like. For example, cell shape change and motility areinvolved at two rate-limiting steps in cancer progression: angiogenesis(i.e., blood vessel recruitment) and metastasis (i.e., spreading of atumor from one location in the body to other locations), in theextravasation of lymphocytes from vascular elements in inflammatorydisease, and in the invasive progression of many cellular parasites intoinfected host tissue. In combination with cell growth inhibitors,treatment with specific cell motility inhibitors has the potential toprovide a more efficacious treatment of diseases of cell motility andproliferation such as inflammatory disease, cancer, infections and thelike, analogous to the multiple drug approach for treatment of HIVinfection and AIDS.

A number of compounds that target actin directly are known to beeffective in modulating cell motility. The best known compounds are thecytochalasins, which are cell-permeable destabilizers of actinfilaments, and phalloidin, which is a cell-impermeable stabilizer ofactin filaments (J. A. Cooper, J. Cell Biol, 105 (1987)). In addition,latrunculins are cell-permeable disrupters of actin filaments (I.Spector, Science, 219, 493 (1983)). Jasplakinolide is a cell-permeablestabilizer of actin filaments (M. R. Bubb et al., Chem., 269, 14869(1994)). A few compounds that target proteins upstream of the actincytoskeleton are known, such as the Rho-kinase inhibitor Y-27632 (M.Uehata et al., Nature, 389, 990 (1997), and myosin light chain kinaseinhibitors, such as ML-g (M. Saitoh et al., Biochem. Biophys, Res.Commun., 140, 280 (1986)). Recently, a cyclic peptide dimer wasdiscovered that inhibits the activity of N-WASP, a protein involved inCdc42-mediated actin nucleation by the Arp2/3 complex (J. R. Peterson etal., Proc. Natl. Acad. Sci. USA, 98, 10624 (2001)). Nevertheless, thereis a dearth of available compounds that affect actin dynamics and cellmotility.

There is thus a widely recognized need and it would be highlyadvantageous to have a ribonuclease of the T2 family having actinbinding activity, that has potential usefulness in the treatment andprevention of cell motility-associated disease such as inflammatorydisease, cancer, neurodegenerative disease and infectious disease.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of preventing, inhibiting and/or reversing proliferation,colonization, differentiation and/or development of abnormallyproliferating cells in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of aribonuclease of the T2 family.

According to another aspect of the present invention there is provided amethod of preventing, inhibiting and/or reversing proliferation,colonization, to differentiation and/or development of abnormallyproliferating cells in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of apolynucleotide encoding and capable of expressing in vivo a recombinantribonuclease of the T2 family.

According to yet another aspect of the present invention there isprovided methods of (i) treating a tumor in a subject; (ii) preventing,inhibiting and/or reversing the development a tumor in a subject; (iii)preventing, inhibiting and/or reversing transformation of a benign tumorto a malignant tumor in a subject; (iv) preventing, inhibiting and/orreversing tumor angiogenesis in a subject; (v) reducing the number ofindividual tumors in a subject; (vi) reducing tumor size in a subject;(vii) reducing a number of malignant tumors in a subject; and (viii)preventing, inhibiting and/or reversing transformation of a tissue intoa tumor in a subject, each of the methods is effected by administeringto the subject a therapeutically effective amount of a ribonuclease ofthe T2 family or a therapeutically effective amount of a polynucleotideencoding and capable of expressing in vivo a recombinant ribonuclease ofthe T2 family.

According to still another aspect of the present invention there isprovided a pharmaceutical composition comprising, as an activeingredient, a ribonuclease of the T2 family, and a pharmaceuticallyacceptable carrier.

According to an additional aspect of the present invention there isprovided a pharmaceutical composition comprising, as an activeingredient, a polynucleotide encoding and capable of expressing in vivoa recombinant ribonuclease of the T2 family, and a pharmaceuticallyacceptable carrier.

According to yet an additional aspect of the present invention there isprovided a method of preparing a medicament useful in preventing,inhibiting and/or reversing proliferation, colonization, differentiationand/or development of abnormally proliferating cells comprising the stepof combining a ribonuclease of the T2 family with a pharmaceuticallyacceptable carrier.

According to still an additional aspect of the present invention thereis provided a method of preparing a medicament useful in preventing,inhibiting and/or reversing proliferation, colonization, differentiationand/or development of abnormally proliferating cells comprising the stepof combining a polynucleotide to encoding and capable of expressing invivo a recombinant ribonuclease of the T2 family with a pharmaceuticallyacceptable carrier.

According to further features in preferred embodiments of the inventiondescribed below, the ribonuclease of the T2 family substantially lacksribonucleolytic activity. As used herein the phrase “substantially lacksribonucleolytic activity” refers to (i) an inactivated ribonuclease(either natural or recombinant) of the T2 family which has 0-10%ribonucleolytic activity as is compared to a similar, non-inactivated,ribonuclease; and/or (ii) a recombinant mutant (natural or man induced)ribonuclease of the T2 family which has 0-10% ribonucleolytic activityas is compared to a similar, non-mutant, ribonuclease. Inactivating theribonucleolytic activity of the ribonuclease of the T2 family may beeffected by a process selected from the group consisting of boiling,autoclaving and chemically denaturing.

According to still further features in the described preferredembodiments the abnormally proliferating cells are cancerous cells.

According to still further features in the described preferredembodiments the step of administering to the subject the therapeuticallyeffective amount of the RNase of the T2 family is effected by anadministration mode selected from the group consisting of oraladministration, topical administration, transmucosal administration,parenteral administration, rectal administration and by inhalation.

According to still further features in the described preferredembodiments the ribonuclease of the T2 family is RNase B1.

According to still further features in the described preferredembodiments the ribonuclease of the ribonuclease T2 family is selectedfrom the group consisting of RNase T2, RNase Rh, RNase M, RNase Trv,RNase hp, RNase Le2, RNase Phyb, RNase LE, RNase MC, RNase CL1, RNaseBsp1, RNase RCL2, RNase Dm, RNase Oy and RNase Tp.

According to still further features in the described preferredembodiments the medicament is identified as providing a treatment for aspecified proliferative disorder or disease, such as a specified cancer.

According to still further features in the described preferredembodiments the abnormally proliferating cells are cell associated witha proliferative disorder or disease selected from the group consistingof papilloma, blastoglioma, Kaposi's to sarcoma, melanoma, lung cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma,head cancer, neck cancer, bladder cancer, breast cancer, colorectalcancer, thyroid cancer, pancreatic cancer, gastric cancer,hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease,Burkitt's disease, arthritis, rheumatoid arthritis, diabeticretinopathy, angiogenesis, restenosis, in-stent restenosis and vasculargraft restenosis, proliferative vitreoretinopathy, chronic inflammatoryproliferative disease, dermatofibroma and psoriasis.

According to yet an additional aspect of the present invention, thereare provided methods of treating or preventing diseases or conditionscharacterized by: (i) excessive cell motility; or (ii) abnormalaccumulation of cells in a subject in need thereof. Each of the methodsis effected by administering to the subject a therapeutically effectiveamount of a ribonuclease of the T2 family or a therapeutically effectiveamount of a polynucleotide encoding and capable of expressing in vivo arecombinant ribonuclease of the T2 family.

According to still further features of the described preferredembodiments, the disease or condition characterized by excessivecellular motility is selected from the group consisting of aninflammatory disease, a neurodegenerative disease and a cancer.

According to yet further features of the described preferredembodiments, the disease or condition characterized by excessivecellular motility is a cancer.

According to further features of the described preferred embodiments,administering to the subject the therapeutically effective amount ofsaid ribonuclease of the T2 family or the expressible polynucleotideencoding the ribonuclease is effected by an administration mode selectedfrom the group consisting of oral administration, intravenousadministration, subcutaneous administration, systemic administration,topical administration, transmucosal administration, parenteraladministration, rectal administration and inhalation.

According to still another aspect of the present invention, there areprovided methods for (i) inhibiting the motility of a cell; (ii)inhibiting actin filament assembly and disassembly in a cell; and (iii)enhancing apoptosis of a cell. Each of the methods is effected byproviding to the cell an effective concentration of a ribonuclease ofthe T2 family or a therapeutically effective amount of a topolynucleotide encoding and capable of expressing in vivo a recombinantribonuclease of the T2 family.

According to further features in the described preferred embodiments,the ribonuclease of the T2 family is a recombinant protein, expressed ina heterologous expression system. The heterologous system can be abacterial expression system, yeast expression system and higher cellexpression system.

According to still further features of the described preferredembodiments, the ribonuclease activity of the ribonuclease protein isthermostable.

According to yet further features of the described preferredembodiments, the ribonuclease of the T2 family is substantially devoidof ribonuclease activity.

According to further features of the described preferred embodiments, aribonuclease activity of the ribonuclease protein is thermostable.

According to still further features of the described preferredembodiments, an actin binding activity of the ribonuclease protein isthermostable.

According to yet further features of the described preferredembodiments, the ribonuclease of the T2 family having actin bindingactivity can be RNase T2, RNase Rh, RNase M, RNase Trv, RNase Up, RNaseLe2, RNase Phyb, RNase LE, RNase MC, RNase CL1, RNase Bsp1, RNase RCL2,RNase Dm, RNase Oy and RNase Tp.

According to further features of the described preferred embodiments,the expressible polynucleotide is selected capable of stable integrationinto a genome of the cell or of a cell of the subject.

According to still further features of the described preferredembodiments, the cell is a cancer cell.

According to still further features of the described preferredembodiments, the providing is effected in vitro or in vivo.

According to still another aspect of the present invention, there areprovided a method of isolating a thermostable ribonuclease of the T2family. The method comprises the steps of: heat denaturating a samplewhich comprises cells expressing a ribonuclease of the T2 family so asto obtain a heat denatured sample, isolating a supernatant of the heatdenatured sample; identifying a fraction of the supernatant to having athermostable ribonuclease of the T2 family; and purifying the fractionhaving the thermostable ribonuclease of the T2 family from thesupernatant to substantial purity.

According to still further features of the described preferredembodiments the heat denaturing is effected by a temperature of at least90° C., for at least 10 minutes.

According to yet further features of the described preferredembodiments, identifying the fraction is effected by monitoringribonucleolytic activity and/or gel electrophoresis.

According to further features of the described preferred embodiments,purifying the fraction is effected by column chromatography.

According to yet another aspect of the present invention there isprovided a method of inactivating a ribonuclease activity, yetmaintaining an actin binding activity of a ribonuclease of the T2family, the method effected by subjecting the ribonuclease to denaturingconditions sufficient for substantially inactivating the ribonucleaseactivity, yet maintaining the actin binding activity.

According to further features of the described preferred embodiments,the inactivating of the ribonuclease activity is effected by autoclavingand/or chemical denaturation.

According to still further features of the described preferredembodiments the ribonuclease is a recombinant ribonuclease.

According to yet further features of the described preferred embodimentsthe ribonuclease is substantially devoid of ribonuclease activity andhas an actin binding activity.

According to a further aspect of the present invention there is provideda method of preparing a medicament useful in treating and/or preventinga disease or condition characterized by excessive cell motilitycomprising combining a ribonuclease of the T2 family having an actinbinding activity, or a polynucleotide encoding and capable of expressingin vivo the ribonuclease of the T2 family having actin binding activity,with a pharmaceutically acceptable carrier.

According to still another aspect of the present invention, there isprovided a method of preparing a medicament useful in treating and/orpreventing a disease or condition characterized by abnormal accumulationof cells, the medicament to combining a ribonuclease of the T2 familyhaving an actin binding activity, or a polynucleotide encoding andcapable of expressing in vivo the ribonuclease of the T2 family, with apharmaceutically acceptable carrier.

According to further features of the described preferred embodiments,the ribonuclease of the T2 family is a recombinant protein, expressed ina heterologous expression system. The heterologous expression system canbe a bacterial expression system, a yeast expression system and a highercell expression system.

According to yet further features of the described preferred embodimentsthe ribonuclease of the T2 family is substantially devoid ofribonuclease activity.

According to still further features of the described preferredembodiments, the ribonuclease can be selected from the group consistingof RNase T2, RNase Rh, RNase M, RNase Trv, RNase Irp, RNase Le2, RNasePhyb, RNase LE, RNase MC, RNase CL1, RNase Bsp1, RNase RCL2, RNase Dm,RNase Oy and RNase Tp.

According to still further features of the described preferredembodiments, the ribonuclease of the T2 family is a recombinant protein,expressed in a heterologous expression system. The heterologousexpression system can be a bacterial expression system, a yeastexpression system and/or a higher cell expression system.

According to yet further features of the described preferredembodiments, the ribonuclease of the T2 family is substantially devoidof ribonuclease activity.

According to still further features of the described preferredembodiments, the ribonuclease of the T2 family having actin bindingactivity is selected from the group consisting of RNase T2, RNase Rh,RNase M, RNase Trv, RNase hp, RNase Le2, RNase Phyb, RNase LE, RNase MC,RNase CL1, RNase Bsp1, RNase RCL2, RNase Dm, RNase Oy and RNase Tp.

According to still another aspect of the present invention, there areprovided methods of enhancing a treatment of a cancer, the enhancingcomprising administering to a subject in need thereof, in combinationwith said treatment of the cancer, a ribonuclease of the T2 familyhaving an actin binding activity, or a polynucleotide encoding andcapable of expressing in vivo said ribonuclease of the T2 family. Thetreatments can be chemotherapy, radiotherapy, phototherapy andphotodynamic therapy, surgery, nutritional therapy, ablative therapy,combined radiotherapy and chemotherapy, brachiotherapy, proton beamtherapy, immunotherapy, cellular therapy and photon beam radiosurgicaltherapy.

The present invention successfully addresses the shortcomings of thepresently known configurations by characterizing novel activities ofribonucleases of the T2 family having actin-binding activity, useful inthe prevention, inhibition and reversal of in the treatment andprevention of cell motility-associated disease such as inflammatorydisease, cancer, neurodegenerative disease and infectious disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a graph representation of the absorbance and RNase activity ofAspergillus niger RNase B1 isolated according to the teachings of Roiz,L. and Shoseyov, O., 1995, Int. J. Plant Sci. 156:37-41. Graph Arepresents fractions obtained via EMD-TMAE column chromatography of acrude filtrate, while graph B represents the fraction obtained fromMONO-Q column chromatography of the active fractions resulted from theEMD-TMAE chromatography of the crude filtrate. The solid line representsabsorbance at 280 nm and the dashed line represents RNase activity.

FIG. 2 is an SDS-PAGE zymogram illustrating the increase in RNase B1protein concentration throughout the purification steps employed. Lane 1represents the crude filtrate; lane 2 represents the eluate from theEMD-TMAE column; lane 3 represents the eluate from the MONO-Q column;lane 4 represent the eluate of lane 3 assayed in situ for RNase activityand stained with toluidine blue; lane 5 represents the purified RNasefollowing deglycosilation by PNGase F. Lanes 1-3 and 5 are stained withcoomassie blue.

FIG. 3 is a graph illustrating the in vitro effect of differentconcentrations of B1 RNase on peach pollen germination (solid line withblack squares) and pollen tube length (dashed line with boxes).

FIGS. 4 a and 4 b illustrate the effect of RNase B1 on peach pollen tubegrowth in the stigma and the upper part of the style. FIG. 4 arepresents the control flower, while FIG. 4 b is a flower treated withRNase B1 before pollination. Bar=0.2 mm

FIGS. 5 a and 5 b illustrate the effect of RNase B1 on pollen tubegrowth in the stigma of a tangerine flower. FIG. 5 a represents acontrol flower which was exposed to open pollination for 48 hours. FIG.5 b represents a flower which was treated with RNase B1 prior topollination. Bar=0.1 mm.

FIGS. 6 a and 6 b illustrate viability test conducted on nectarineseeds. FIG. 6 a represents a control seed produced by an untreatedflower, while FIG. 6 b represents a seed produced by an RNase B1 treatedflower. Bar=0.3 mm.

FIG. 7 illustrates the effect of RNase B1, untreated, boiled orautoclaved, on lily cv. Osnat pollen tube length.

FIGS. 8 a and 8 b illustrate the effect of RNase B1 on lily pollen tubesgrowing in vitro and stained with IKI.

FIGS. 9 a and 9 b illustrate still shots captured from integrated videoimages showing organelle movement and localization in RNase B1 untreated(FIG. 9 a) and treated (FIG. 9 b) pollen tubes.

FIGS. 10 a and 10 b illustrate the effect of RNase B1 on actin filamentsof a growing lily pollen tube. FIG. 10 a represents the control pollentube whereas FIG. 10 b represents the RNase B1 treated pollen tube. Bothpollen tubes were excised and stained with TRITC phalloidine forvisualization following experimentation.

FIG. 11 is a Scatchard plot representing RNase B1 binding to actin.A-actin concentration (μM), Rf-free RNase B1 concentration (μM),Rb-bound RNase B1 concentration (μM).

FIGS. 12 a-c illustrate immunogold silver stained lily pollen tubesgrown for 1 hour. FIG. 12 a represents a control, whereas FIGS. 12 b and12 c are both RNase to B1 treated pollen tubes. The pollen tube of FIG.12 b was incubated with rabbit pre-immune serum, while the pollen tubeof FIG. 12 c was incubated with anti-RNase B1 rabbit polyclonalantibody.

FIGS. 13 a and 13 b illustrate the effect of different concentrations ofRNase B1 on the viability of HT29 colon cancer cells. Replicate samplesof cells were grown for 48 hours or for 72 hours at 37° C., visualizedusing trypan blue differential staining and counted. FIG. 13 arepresents the total numbers of cells whereas FIG. 13 b represents thepercent of dead cells.

FIG. 14 illustrates the effect of RNase B1 on clonogenicity of HT29cells. Replicate samples of cells were preincubated with growth mediumin the absence or presence of 10⁻⁶ M RNase B1 for 48 hours, trypsinized,washed, resuspended in RNase B1-free growth medium in serial dilutions,and plated into 96-well microtiter plates to colonize for 14 days.Colonies were counted following fixation and staining with methyleneblue.

FIG. 15 illustrates the effect of exposure period to RNase B1 on theclonogenicity of HT29 cells. Replicate samples of cells werepreincubated with growth medium containing 10⁻⁶ M RNase B1 for 48 hoursand than let to colonize in growth medium containing the sameconcentration of RNase B1, or in RNase B1-free medium. Colonization wasdone in 96-well microtiter plates for 7 days. Each treatment containeddifferent initial numbers of cells per well. The colonies were countedfollowing fixation and visualization in methylene blue. Cellspreincubated and colonized in RNase B1-free growth medium served as acontrol.

FIGS. 16 a-c illustrate the effect of RNase B1 on the colonizationability of HT29 cells. Control cells (FIG. 16 a) were preincubated 48hours in RNase B1-free growth medium and then trypsinized and incubatedwith the same growth medium in 96-microtiter plates for colonization.FIG. 16 b represents cells that were preincubated for 48 hours in growthmedium containing 10⁻⁶ M RNase B1 and then allowed to colonize in RNaseB1-free growth medium. FIG. 16 c represents cells that were preincubatedand then colonized in growth medium containing 10⁻⁶ M RNase B1. The cellcolonies were visualized using methylene blue staining.

FIG. 17 is a scheme of in vivo experiments conducted in rats, describingthe treatment for each group of 6 rats.

FIG. 18 demonstrates the effect of two different pHs on the rate ofRNase B1 release from CAP microcapsules. Microcapsules containing 10 mgRNase B1 were suspended in 10 ml of 0.1 M HCl (pH 1) or 0.1 M Trisbuffer (pH 8) and incubated at 37° C. while stirring. Samples of uppersolution were taken every 30 min for RNase activity tests.

FIGS. 19 a-d demonstrate the effect of RNase B1 and/or DMH on ratsgrowth rate, as shown by body weigh at the end of each experiment.Initial rat weight was about 200 grams. n=6. 19 a—PBS, RNase B1 orI-RNase B1 was given via osmotic pumps at weeks 1-9 after first DMHinjection (preventive treatment). As control rats treated as describedabove, but in the absence of DMH were used. 19 b—PBS, RNase B1 orI-RNase B1 was given via osmotic pumps at weeks 12-17 after first DMHinjection (therapeutic treatments). 19 c—The rats were fed withmicrocapsules containing RNase B1 or glucose as a preventive treatment.As control rats that were treated with RNase B1 in the absence of DMHwere used. 19 d—Rats were fed with microcapsules containing RNase B1 orglucose.

FIGS. 20 a-c demonstrate RNase activity in feces of rats implanted withosmotic pumps containing RNase B1 (20 a), I-RNase B1 (20 b) or PBS (20c), as a preventive treatment. As control, rats were treated with RNaseB1 or PBS in the absence of DMH. RNase activity was determined asdescribed in the Examples section below.

FIG. 21 demonstrates RNase activity in feces of rats fed withmicrocapsules containing RNase B1 or glucose as a preventive treatment.As control, rats were fed with RNase B1 or glucose in the absence ofDMH. RNase activity was determined as described in the Examples sectionbelow.

FIG. 22 show the number of aberrant crypt foci (ACF) in distal colon (5cm) of rats implanted with osmotic pumps as a preventive treatment(n=6).

FIGS. 23 a-c demonstrate the effect of RNase B1 on different parametersexamined in the distal (5 cm) colon of rats fed with microencapsulatedRNase B1 or glucose, as a preventive treatment (n=6). 23 a—number oftumors per colon; 23 b—tumor size; 23 c—ACF per colon.

FIGS. 24 a-d demonstrate different types of tumors, as photographed atthe to inner mucosal surface 1 hour after excision. 24 a—red tumors; 24b—white tumors. 24 c—a pink tumor and a red tumor; 24 d—distribution ofthree types of tumors in rats fed with microcapsules containing glucoseor RNase B1, as a preventive treatment.

FIGS. 25 a-d show histopathological examination of tumors stained withMayer's heamatoxylin and martius-yellow. 25 a—an adenoma oradenopapilloma—a benign tumor; 25 b—adenocarcinoma, in which mucosalcells penetrated beneath the sub-mucosa; 25 c—a well-developedadenocarcinoma, in which tissue arrangement is entirely interrupted; 25d—distribution pattern of adenoma and adenocarcinoma types of tumors incolons of rat treated with encapsulated glucose or RNase B1, as apreventive treatment.

FIGS. 26 a-c demonstrate the effect of RNase B1 on different parametersexamined in the distal (5 cm) colon of rats treated with osmotic pumpscontaining PBS, RNase B1 or I-RNase B1 as a therapeutic treatment. 26a—number of tumors per colon; 26 b—distribution of tumors according tosize; 26 c—distribution of tumors according to color, indicatingangiogenesis.

FIGS. 27 a-c demonstrate the effect of RNase B1 on different parametersexamined in the distal (5 cm) colon of rats fed with microencapsulatedRNase B1 or glucose as therapeutic treatments. 27 a—number of tumors percolon; 27 b—distribution of tumors according to size; 27 c—distributionof tumors according to color, indicating angiogenesis.

FIGS. 28 a-b show human colon carcinoma HT-29 4-d cultured cells stainedwith TRIRC for actin. 28 a—control cells; 28 b—cells that were grown inthe presence of 10⁻⁶ M RNase B1.

FIGS. 29 a-b show human colon carcinoma HT-29 4-d cultured cellsimmunostained for membranal actin. 29 a—control cells; 29 b—cells grownin the presence of 10⁻⁶ M RNase B1.

FIGS. 30 a-c show human colon carcinoma HT-29 4-d cultured cellsimmunostained with FITC. Anti-RNase B1 was used as the primary antibody.30 a—control cells; 30 b—cell that were grown in the presence of RNaseB1, showing RNase B1 bound on the cell surface; 30 c—pre immuned serum(PIS) was used as primary antibody.

FIG. 31 demonstrates the effect of different protein treatments on lilypollen to tube length. Pollen tubes were grown in vitro for 1 hour at25° C. as described in the Examples section that follows.

FIGS. 32 a-h are photomicrographs illustrating HUVEC tube formation onMatrigel in the presence or absence of 1 μg/ml angiogenin, and in thepresence or absence of RNases (2 μM each). FIG. 32 a—Absence ofangiogenin and RNase (Control); FIG. 32 b—Angiogenin (Positive Control);FIG. 32 c—RNase B1 (Negative Control); FIG. 32 d—RNase B1+angiogenin;FIG. 32 e—RNase T2 (Negative Control); FIG. 32 f—RNase T2+angiogenin;FIG. 32 g—RNase I (Negative Control); FIG. 32 h—RNase I+angiogenin Notethe superior inhibition of endothelial tube formation by Aspergillusniger RNase B1 (FIG. 32 d), as compared to Aspergillus oryzae T2 RNase(FIG. 32 f).

FIGS. 33 a-d are photographs illustrating the effect of RNase B1 onB16F1 melanoma in an ip/ip experiment in BDF1 (black) and Balb/c (white)mice. Mice were intraperitoneally (i.p.) injected with 2×10⁶ B16F1melanoma cells and following 24 hours and 5 days the mice were furtheri.p. injected with either RNase B1 (5 mg/mouse in 100 μl PBS) or PBS.The presence of tumors was evaluated 14 days following melanoma cellinjection. FIG. 33 a—Balb/c injected with melanoma cells only; FIG. 33b—Balb/c injected with melanoma cells and RNase B1; FIG. 33 c—BDF1injected with melanoma cells only; FIG. 33 d—BDF1 injected with melanomacells and RNase B1. Note the highly developed typical black tumors inthe melanoma mouse models (FIGS. 33 a and c, arrows), compared withconsiderably smaller tumors in RNase B1-treated mice (FIGS. 33 b and d,arrows).

FIGS. 34 a-e illustrate the effect of RNase B1 in reducing thedevelopment of B16F10 melanoma foci formed in the lungs of balb/c mice,implanted with 5×10⁵ (FIGS. 34 a-c) or 5×10⁶ (FIGS. 34 d-e) cells/mouse.FIGS. 34 a-b are photomicrographs of the lungs of the B16F10-implantedmice following RNase B1 (10 mg/mouse, three times, every four days,starting 24 hours after cells injection) or PBS treatment. FIG. 34a—PBS; FIG. 34 b—RNase B1. Note the significant decrease in melanomafoci and metastases following RNase B1 treatment (FIG. 34 b, arrows) ascompared to PBS injection (FIG. 34 a, arrows). FIGS. 34 c-e are graphsdepicting a quantitative determination of the number of metastases perlung (FIG. 34 c), the lung weight (FIG. 34 d) and the mean metastasisdiameter (FIG. 34 e). Note the significant decrease in the number ofmetastases/lung (76%, P<0.001, FIG. 34 c), the average lung weight (25%,P<0.01, FIG. 34 d), and the mean metastasis diameter (25%, P<0.001, FIG.34 e) in the RNase B1-treated mice as compared with PBS injected mice.

FIG. 35 illustrates the effect of RNase B1 on tumor growth of humanmelanoma cells in nude mice. A375SM melanoma cells (5×10⁵) were injectedsubcutaneously into nude mice (n=5) and three days later, animalsinjected with the tumor cells were subsequently intraperitoneallyinjected with either 1 mg of RNase B1/mice or PBS (control) Animals wereinjected for 30 days (every other day, 3 days a week) and the tumordimensions were recorded at the noted times. Shown are tumor volumes asexpressed in mm³ as calculated from the equation of (length×width)/2.Note that while in mice injected with PBS the tumor volume drasticallyincreased and reached the size of 800 mm³ within 30 day, in RNaseB1-treated mice the tumor volume remained relatively low and did notexceed 180 mm³ at thirty days-post melanoma cells injections.

FIGS. 36 a-b illustrate the effect of RNase B1 on tumor size in HT-29derived nude mice xenografts in an s.c./i.v. experiment. Tumors wereinduced in mice using HT-29 cells (3×10⁶ per mouse) and the mice werefurther subjected to RNase B1 (2 injections containing 5 mg RNase B1each in 100 μl PBS) or PBS (control) injections. FIG. 36 a—an overallview of tumors in PBS (control, yellow arrow) or RNase B1 (bluearrow)-treated. Note the significant decrease in the size of tumor inthe RNase B1-treated mice (blue arrow) as compared with the PBS injectedmice (yellow arrow); FIG. 36 b—is a bar graph depicting tumor size inPBS or RNase B1-treated mice (n=5). Note the average 60% decrease intumor size following RNase B1 treatment.

FIG. 37 is a photograph illustrating in vivo inhibition of angiogenesisby RNase B1. Gel foams impregnated with 100 ng/sponge angiogenin weresubcutaneously implanted in both sides of a nude mouse and following 48hours the mice were intraperitoneally injected at both sides of theperitoneaum (7 times, every two days), each with a different treatment;one side with RNase B1 (250 μM RNase B1 in 100 μl) and the other sidewith PBS. Note the significant angiogenesis in the side of PBS injection(circled region with an arrow, Angiogenin) as compared with thenegligible angiogenesis in the side of RNase B1 injection (circledregion with an arrow, Angiogenin and RNase B1), indicating RNaseB1-mediated inhibition of the angiogenin-induced development of bloodvessels.

FIG. 38 is a bar graph illustrating the effect of RNase B1 in inhibitingA375SM cell invasion. A375SM cells were treated in the presence orabsence of RNase B1 and following 22 hours the number of cells invadedthe Matrigel-coated filter were counted. Note the dose-dependent effectof RNase B1 in inhibiting A375SM invasion to the Matrigel-coated filter.While in untreated A375SM cells (control) 1216±68 cells penetrated theMatrigel-coated filters, 725±59 or 211±14 A375SM cells which weretreated with 1 or 10 μM RNase B1 penetrated the Matrigel-coated filters.*=P<0.01; **=P<0.001.

FIGS. 39 a-b are bar graphs illustrating HT-29-derived nude micexenografts in the sc/ip model. RNase B1 was injected at the notedconcentrations starting from 24 hours following cells implantation (10⁶cell/mouse) and every other day. Tumor size was measured 30 daysfollowing the onset of RNase B1 treatment. Note the significantreduction (up to 60%) in tumor size following RNase B1 treatment at awide range of doses, 0.001-1 mg/injection (FIG. 39 a) and 1-5mg/injection (FIG. 39 b) in the two subsequent experiments (N=6 in eachcase), demonstrating the preventive (FIG. 39 a) and therapuetic (FIG. 39b) effects of RNase B1 treatment. Cont.=HT-29-implanted mice which wereinjected with PBS.

FIGS. 40 a-c are photomicrographs of peritoneum cross sections fromHT-29-implanted nude mice in the s.c./i.p. model in the presence orabsence of RNase B1. FIG. 40 a—Hematoxylin and Eosin (H&E) staining.Note the thin peritoneum (arrow) overlaying the muscle; FIGS. 40b-c—Immunostaining of peritoneum taken from an RNase B1-treated mouse(FIG. 40 b) or PBS-treated mouse (FIG. 40 c), using rabbit anti-RNase B1and FITC-conjugated goat anti rabbit. Note the green fluorescencedemonstrating the accumulation of RNase B1 onto the peritoneum of theRNase B1-treated mouse but not the PBS-treated mouse.

FIGS. 41 a-c are photomicrographs of cross sections from HT-29-derivedtumors of the s.c./i.p nude mice model. FIG. 41 a-H&E staining; FIGS. 41b-c—Immunostaining of a blood vessel of a tumor derived from an RNaseB1-treated (FIG. 41 b) or PBS-treated (FIG. 41 c) mouse, using rabbitanti-RNase B1 and to FITC-conjugated goat anti-rabbit. Arrows indicateblood vessels. Note the accumulation of RNase B1 onto the basalmembrane.

FIGS. 42 a-b are representative histopathology sections of colon cancertissue illustrating the effect of RNase B1 on CD31 expression onDMH-induced colonic tumors. Tumor sections from dimethylhydrazine(DMH)-treated rats (FIG. 42 a) or from DMH and RNase B1-treated rats(FIG. 42 b) were stained with an anti-CD31 antibody (sc-8306 antibodySanta Cruz Biotechnology Inc. Santa Cruz, Calif.). Note the presence ofhigh number of large blood vessels in the tumors of DMH-treated rats(FIG. 42 a) and the significantly lower size and number of blood vesselsin DMH-RNase B1-treated rats (FIG. 42 b). Magnification×200. Sizebar=100 μm.

FIGS. 43 a-b are photomicrographs of representative histopathologysections of tumors in nude mice showing the pro-apoptotic effect ofRNase B1 on tumor size and apoptosis in mouse melanoma cells. MouseB16F1-derived melanoma cells (2×10⁶ cells/mouse) were grown in theintraperitonal cavity of BDF1 and Balb/c mice. RNase B1-treated micereceived two intraperitoneal injections of 5 mg RNase B1 in 100 μl PBS,24 hours and 5 days after injection with the melanoma cells. Untreatedcontrol mice received PBS injections. After 14 days, the mice weresacrificed and samples prepared for analysis. Paraffin cross-sectionswere stained for apoptosis using Klenow-FragEl kit (Oncogene, Cambridge,Mass.). Note the predominance of apoptotic cell nuclei (brown stain) inthe tumors from RNase B1-treated mice (FIG. 43 b), as compared with theactively dividing nuclei (green stain) in the untreated controls (FIG.43 a).

FIGS. 44 a-b are photomicrographs of representative colon tumor sectionsillustrating the effect of RNase B1 on apoptosis rate in DMH-inducedcolonic tumors. Sections of colon tumors from dimethylhydrazine(DMH)-treated rats were fixed and embedded in paraffin, and assayed forapoptosis by the deoxynucleotide transferase-mediated dUTP-nickend-labeling (TUNEL) assay using the Klenow-FragEl (Oncogene, Cambridge,Mass.). Apoptotic cells were stained and visualized (brown staining)with peroxidase-conjugated antidigoxigenin antibodies. FIG. 44a—DMH-treated rat; FIG. 44 b—RNase B1-DMH-treated rat. Note that whileno TUNEL-positive cells are observed in tumor sections of DMH-treatedrats (FIG. 44 a), a large to number of TUNEL-positive (i.e., apoptotic)cells are observed in tumor sections of RNase B1-DMH-treated rat (FIG.44 b). Size bar=100 μm.

FIGS. 45 a-d are representative histopathology sections of tumors innude mice showing the pro-apoptotic effect of RNase B1 on tumor size andapoptosis in human colon cancer xenografts. HT29 cancer cells (2.5×10⁶cells/mouse) were injected into the left hip of nude mice. RNaseB1-treated mice (n=5) received 3 intravenous injections of 5 mg RNase B1every 5 days, starting 24 hours following cancer cells injection (FIGS.45 b and d). Control mice (n=5) were left untreated (FIG. 45 a and c).Paraffin sections of tumors were stained by Hematoxylin and Eosin (H&E)for histology evaluation (FIGS. 45 a-b) and with Klenow-FragEl kit(Oncogene, Cambridge, Mass., FIGS. 45 c-d) for apoptosis. Vital andactively dividing nuclei are stained bright green, whereas apoptoticnuclei are stained brown. Note the condensed cytoplasm and nuclei (FIG.45 b) and high proportion of apoptotic cells (FIG. 45 d) in the tumorsfrom RNase B1 treated mice, as compared with the normal vital appearance(FIG. 45 a) mitotic activity (FIG. 45 c) in the tumors from untreatedcontrols.

FIG. 46 is a graph illustrating the effects of Taxol and RNase B1 onrelative volume of LS174T-induced tumors. Balb/c (athymic mice (CD-1nu/nu; Charles River, Wilmington, Mass.) nude mice were s.c. injectedwith LS174T cancer cells and 10-13 days following cell injection (whentumors were palpable) the mice were treated with i.p. injections of thenoted treatments for 5 consecutive days out of 7 days over a period of 3weeks. Control: PBS or propylene glycol+ethanol; RNase B1: 50 mg/kgRNase B1 (1000 μg/injection); RNase B1+Taxol: 50 mg/kg RNase B1 and 5mg/kg Taxol; Taxol: 5 mg/kg Taxol (100 μg/injection). Note thesignificant effect on the relative tumor volume (RTV) in the RNaseB1-treated mice as compared to Taxol-treated mice, and the unpredictedsignificant inhibition of tumor growth in mice treated with the combinedtreatment of RNase B1 and Taxol.

FIGS. 47 a-b are Western blot (FIG. 47 a) and SDS-PAGE protein staining(FIG. 47 b) of actin and actin-binding proteins illustrating the abilityof RNase B1 to bind actin in vitro. Actin and actin-binding proteinswere run on an SDS-PAGE and transferred on a nitrocellulose membrane.The membrane was incubated with actin and then with anti-actin followedwith peroxidase-conjugated goat anti mouse IgM to [Actin (Ab-1) Kit, CAT#CP01-1EA (Oncogene)]. Signals were detected using the ECL detectionsystem (Pierce CAT No #24080). Lane 1—RNase B1, lane 2—Angiogenin, lane3—E. coli RNase I, lane 4—actin. Note the strong band intensity in lanes1 and 4 indicating the strong association of RNase B1 to actin.

FIG. 48 is a bar graph illustrating a dose-dependent effect of RNase B1in inhibiting total MMP-2 release by A375SM melanoma cancer cells.A375SM cells were grown in Complete Eagle's minimum essential medium(CMEM) in the presence or absence of 1 or 10 μM RNase B1 and the levelof MMP-2 in the medium was measured using ELISA [Quantikine MMP-2immunoassay kit (R&D Systems Inc., Minneapolis, Minn.)] and is expressedas mg MMP-2/10⁶ cells. Note the maximal inhibitory effect obtained inthe presence of 10 μM RNase B1.

FIGS. 49 a-b are zymograms of MMP-2 Collagenase activity illustratingthe effect of RNase B1 treatment on MMP-2 release in A375SM (FIG. 49 a)or HUVEC (FIG. 49 b) cells. The supernatant of cells treated in thepresence or absence of various concentrations of RNase B1 was loaded ongelatin-containing SDS gels and following electrophoresis the gels wereTriton-treated and stained with Coomassie Blue. The presence of MMP-2Collagenase activity is seen as a white band on the blue gel reflectinggelatin-degradation by the 72 kDa MMP-2 collagenase activity. Note theintense bands in A375SM cells in the absence (FIG. 49 a, control) orpresence of 1 μM RNase B1 and the significant decrease in band intensityfollowing treatment of A375SM cells with 5 and 10 μM RNase B1 (FIG. 49a). Also note the dose-dependent decrease in band intensity of the 72kDa MMP-2 in the presence of 1 and 10 μM RNase B1 in HUVECs cells (FIG.49 b), as well as the decrease in band intensity of the MMP-9 (FIG. 49b, arrow); CMEM (Complete Eagle's minimum essential medium)-mediacontaining sera=positive control.

FIGS. 50 a-s are confocal photomicrographs illustrating the cellularlocalization of RNase B1 in RNase B1-treated HUVEC cells. HUVEC cellswere treated with 0.4 mg/ml (i.e., 10 μM) RNase B1 and following 48hours the cells were subjected to immunostaining using the RNase B1 andCD31 antibodies. Shown are the results of serial digital foci done bythe confocal microscope. All slices were done at the same time point andshow different areas in the treated cells. CD31=red, RNase B1=green.Note that the RNase B1 gradually penetrates the cell membrane (asdetected by to the red label of CD31 which is expressed only in themembrane of HUVEC cells) and enters into the cell.

FIGS. 51 a-c are confocal photomicrographs illustrating the cellularlocalization of RNase B1 in RNase B1-treated A375SM cells. A375SM cellswere treated with 0.4 mg/ml (i.e., 10 μM) RNase B1 and the cellularlocalization of the RNase B1 was detected following 2 (FIG. 51 a), 4(FIG. 51 b) or 8 (FIG. 51 c) hours using RNase B1 immunostaining andconfocal microscopy. Note the gradual penetration of RNase B1 into thecell membrane (FIG. 51 a, 2 hours), cell cytoplasm (FIG. 51 b, 4 hours)and cell nuclei (FIG. 51 c, 8 hours). Also note the RNase B1-inducedrounding of the cells following 4 hours (FIG. 51 b) and the apoptoticcharacteristics of the A375SM melanoma cells following 8 hours (FIG. 51c).

FIG. 52 is an agarose gel image illustrating the amplification of a T2RNase from A. niger genomic DNA. Genomic DNA prepared from A. niger wassubjected to PCR amplification using the forward(5′-TTYTGGGARCAYGARTGGAAY-3′, SEQ ID NO.:1, for amino acids F107-N112)and reverse (5′-CCYTTIACRTTRAARTARTARTA-3′, SEQ ID NO.:2, for aminoacids Y200-K206)] degenerate PCR primers designed according to aminoacids F107-N112 and Y200-K206 found identical in RNase B1 and A. saitoiRNase M (GenBank Accession No. P19791, SEQ ID NO.:3). Note the presenceof a 400 bp PCR product reflecting A. niger T2 RNase coding sequence.

FIG. 53 is the nucleotide sequence of the 300 bp PCR product (SEQ IDNO.:4) prepared from the A. niger genomic DNA (as in FIG. 52). Note theopen reading frame of the amino acid sequence (SEQ ID NO.:5) which isidentical to F107-K206 of RNase M, except that in RNase B1 E123 (boxed)replaces D123 of A. saitoi RNase M.

FIG. 54 is silver stain analysis of purified human recombinant RNase6PL.The recombinant protein of the RNase-positive yeast colony was purifiedby heat denaturation, centrifuged, and passed through a Q Sepharosecolumn in a Fast FPLC and the eluted protein was loaded on an SDS-PAGE.Note the obtained 27 kDa purified protein (Lane 1). Lane 2: proteinmolecular markers.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of the use of a ribonuclease of the T2 familyor polynucleotide encoding same for modulating cellular motility,thereby preventing, inhibiting and/or reversing proliferation,colonization, differentiation, development of abnormally proliferatingcells, inflammation, and/or infection in a subject. The presentinvention is further of pharmaceutical compositions containing, as anactive ingredient, the ribonuclease of the T2 family having actinbinding activity or a polynucleotide encoding same for treating diseasesor disorders of cell motility in general, and inflammation,neurodegenerative disorders, cellular parasites and cancer inparticular.

The use of ribonucleases with cytotoxic activity for inhibiting theproliferation of tumor cells is not new and has been demonstratedpreviously in the art. A ribonuclease of the A family which iscommercially known as ONCONASE has been shown to inhibit cellproliferation in tumorous tissue in clinical trials. Several otherRNases of the RNase A superfamily have also been demonstrated to havecytotoxic activity in addition to their ribonucleolytic activity. Thetoxicity of ONCONASE and other cytotoxic RNase A variants has been shownto depend on their ability to evade the cytosolic ribonuclease inhibitorprotein (RI) and degrade cellular RNA (Haigis et al., Nuc Acid Res 2003;31:1024-33).

Although the cytotoxicity of some ribonucleases is dependent to someextent on their ribonucleolytic activity, the level of ribonucleolyticactivity does not always correlate to the level of cytotoxicity observedfor ribonucleases. Furthermore, there exist several examples ofribonucleases which do not display cytotoxic activity altogether, yetfunction well as ribonucleases. The most known example is RNase A. Inother cases, the reaction rate is sacrificed for more specific bindingor improved function in some other capacity. For example, angigenin'sactive site is blocked by side chains that are not present in RNase A,rendering it 10.000-fold less active on general substrates, but morespecific in cleaving ribosomal RNA. BS-RNase is a faster nuclease whenit is monomeric. However, its cytotoxicity is greater, and inhibition byribonuclease inhibitor is considerably reduced in the dimer form.Glycosylated RNase B is less active than RNase A on most substrates,while a frequently observed deamidation in BS-RNase, (asparagine 67 toisoaspartate) reduces the activity of RNase A mutants by cleaving awhole chain of H-bond structures in the to protein. (reviewed in Shein,C. H. 1997. Nature Biotechnol 15: 529-536).

Ribonucleases of the T2 family are characterized by their uniquemolecular features. A comparison between RNase members of the A and ofthe T2 families is summarized below in Table 2 (Location of amino acidsare after RNase A and RNase T2 in families A and T2, respectively).

TABLE 2 Feature RNase A RNase T2 Molecular mass 11-14 kDa 36 kDa. (withthe exception of BS-RNase) Optimal 37° C. 50-60° C. temperature forRNase activity: Optimal pH for 6.5-8 3.5-5 RNase activity:Glycosilation: Not glycosylated 12-25% of the total molecular mass Basespecificity: Pyrimidine base-specific. Non specific with adenylic acidpreferential. Disulfide bonds: Four: Five: Common: Cys28-84, Cys40-96,Cys58- Cys3-20, Cys10-53, Cys19-120, 110. Cys63-112 and Cys182-213. Inpancreatic RNases the fourth S-S bond is located between Cys65-72,forming a loop containing Glu69 and Asn71, which are part thenucleotide-binding site. In ONCONASE and bullfrog lectin Cys87- Cys104form a COOH-terminal loop, which is located near the active site.Angiogenins have only 3 disulfide bonds. Mechanis of RNase Active siteActive site activity: Two steps in RNA cleavage RNA catalysis is similarto RNase (i) His12 acts as a general base and A. His46 and His109function as removes a proton from the 2′-hydroxyl general acid and basecatalysts. group of the RNA. His119 acts as a Glu105 and Lys108 mightplays a general acid, donating a proton from the 5′ role in polarizingthe P═O bond O of the leaving nucleotide. of the substrate or instabilizing (ii) The resultant 2′3′-cyclic nucleotides the pentacovalenttransition state. are hydrolyzed, with the roles of His12 Substratebinding sites: and His119 reversed. Lys41 stabilizes the His104 (Inplants it is Tyr or Asp) pentavalent transition state. might act as thephosphate Substrate binding sites: receptor of the substrate. GLn11 andPhe120 form hydrogen bonds There are two recognition sites: with thesubstrate. The major (B1) site contains In ONCONASE and bullfrog lectineGlu11 Tyr57, Trp49 and Asp51. Asp51 forms H-bond with the phosphate ofthe is responsible for the adenine substrate. base recognition. Gln96,Asn71, Glu111, of which Asn71 is A minor (B2) site contains the mostconserved, might catalyze RNA Phe101, Gln95, Asn94, Ser93, cleavage.Pro92 and Gln32.

The ribonucleases of the T2 family have been identified in numerousmicroorganisms, as well as in plants, in which they play an active rolein the pollination process, by selectively limiting the elongation ofpollen tubes racing towards the ovules.

As uncovered by the inventors of the present invention and as is furtherdetailed hereinbelow in Examples 1, 2 and 6, RNase B1, a T2ribonuclease, either ribonucleolytically active or ribonucleolyticallynon-active, specifically binds to actin in elongating pollen tubes tothereby inhibit the elongation of pollen tubes and also to to actin ofmammalian cells.

Actin is known to form filaments which are essential cytoskeletalcomponents of cells, active in both maintaining cellular structure andin supporting intracellular transport of organelles. As a result, actinfilaments are crucial to many cellular processes throughout the lifecycle of normal and abnormal cells, including the motility,proliferation, colonization, differentiation, transformation, andsurvival of a variety of cells of mesodermal and/or neuroectodermaland/or ectodermal and/or endodermal origin, including fibroblasts, cellsof the immune system, cells of the nervous system, cardiac muscle cells,skeletal muscle cells, vascular endothelial cells, vascular smoothmuscle endothelial cells, and cells involved with repair of tissueinjury and other developmental aspects including tissue formation.Numerous studies have shown that actin also participates in variouscellular processes controlling generation of cancer cells (Jordan, M. A.& Wilson, L. 1998. Curr. Opin. Cell Biol. 10:123-130; Jammy, P. A. &Chaponnier, C. 1995. Curr. Opin. Cell Biol. 7:111-117: Sigmond, S. H.1996. Cum Opin. Cell Biol. 8:66-73; Tapon, N. et al. 1997. Cum Opin.Cell Biol. 9:86-92). Thus, for example, actin filaments participates inabnormal cell proliferation (Assoian, R. K. & Zhu, X. 1997. Curr. Opin.Cell Biol. 9:93-98). Malignant cells were found more sensitive tocytochalasin B than normal cells (Hemstreet G. P. et al. 1996. J. CellBiochem. 25S:197-204).

Since actin is a highly conserved protein, maintaining a high level ofhomology between evolutionary distant organisms it was hypothesized thatthe actin binding activity of RNase B1, which inhibits pollen tubeelongation can be utilized, to without being limited by this theory, tospecifically bind actin of mammalian cells, to thereby inhibit theproliferation, colonization, differentiation and/or development thereof.

While reducing the present invention to practice and as is furtherdescribed in Example 2 and 5 of the Examples section, exogenous RNase B1specifically binds to membranal actin and causes a cellular actinnetwork disorder. As is shown in Examples 3-5, the effect of RNase B1 onmammalian cancer cells was further investigated in vitro and in vivo. Asclearly demonstrated therein, RNase B1 (i) substantially decreasesproliferation and/or colonization of adenocarcinoma cells grown inculture; and (ii) reduces the number of aberrant crypt foci (ACF),reduces the number and size of tumors, interferes with tumorangiogenesis, reduces the malignancy of tumors and the transition fromadenoma to adenocarcinoma in a colon carcinoma rat model, in apreventive and/or therapeutic manner, while having no apparent sideeffects on healthy tissue in the colon or elsewhere.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or illustrated bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting. Also, it is to beunderstood that the present invention is not bound to, or limited by,any theory or hypothesis which is indicated herein.

One or more ribonucleases of the T2 family are collectively referred toherein as T2-RNase. Similarly, one or more polynucleotides encoding oneor more ribonucleases of the T2 family are collectively referred toherein as a polynucleotide encoding a T2-RNase (or same).

While reducing the present invention to practice, the inventors haverevealed a previously undisclosed actin binding activity of members ofthe T2 RNase family, separate and independent of the ribonucleolyticcatalytic activity of the proteins (see Example 6 and FIG. 31,hereinbelow). Actin binding activity of the T2 RNase has been quantifiedin vitro (see Example 3, FIG. 11 hereinbelow), and has been localized tothe cell surface in diverse models of cell growth, motility andproliferation (see Example 3, FIGS. 12 a-12 c, and Example 5, FIGS. 29and 30 hereinbelow). Further, the T2 RNase actin binding activitycorrelates with disruption of actin filament organization in cellprotrusions, and strong inhibition of tube growth (see Example 3, FIGS.9 a and 9 b, and Example 5, FIGS. 28 a and 28 b hereinbelow) andangiogenesis (see Example 4, FIGS. 24 a-24 c and 26 c hereinbelow).Further results revealed that the T2 RNase actin binding activity,disruption of cell protrusions and inhibition of motility furthercorrelates with significant inhibition of tumor growth (see Examples 4,FIGS. 22-24 hereinbelow) and prevention of malignancy (see Example 4,FIG. 26 hereinbelow) in both newly proliferating and established,well-developed tumors. Microhistopathological and DNA-based (TUNEL)analyses of the T2 RNase-treated tumors has revealed a strongproapoptotic effect of T2 RNase having an actin binding activity,associated with inhibition of tumor proliferation and metastatic growth,and reduction in tumor volume (see Example 14, FIGS. 43 a-43 b, 44 a-44b and 45 a-45 b hereinbelow). As detailed in the Background sectionhereinabove, dynamic actin filament assembly and disassembly at the cellsurface is highly regulated and is critical to all aspects of cellmotility, growth and development of both normal and abnormal cells.

Thus, according to one aspect of the present invention there is provideda method of preventing, inhibiting and/or reversing proliferation,colonization, differentiation and/or development of abnormallyproliferating cells in a subject. The method according to this aspect ofthe present invention is effected by administering to the subject atherapeutically effective amount of a ribonuclease of the T2 family orof a polynucleotide encoding and capable of expressing in vivo arecombinant ribonuclease of the T2 family, either per se or as an activeingredient of a pharmaceutical composition.

While reducing the present invention to practice, it was uncovered, forthe first time, that exposing abnormally proliferating cells, such astumors, xenografts and metastases (see Example 10 and 14 and FIGS.43-45,) to a ribonuclease of the T2 family, enhanced apoptotic processesin the cells. The proapoptotic effects of the T2 RNase correlated withthe suppression of tumor and metastatic growth and proliferation seen inExamples 10 and 14. It will be appreciated, that diseases associatedwith inhibition of apoptosis include those diseases in which anexcessive accumulation of cells occurs (neoplastic diseases, autoimmunediseases).

In both solid and haematological tumors, the malignant cells show anabnormal response to apoptosis inducers (Watson A J M. 1995. Gut 37:165-167; Burch W. et al. 1992. Trends Pharmacol Sci 13:245-251). Inthese diseases cycle-regulating genes such as p53, ras, c-myc and bcl-2suffer mutations, inactivation or dysregulations associated to malignantdegeneration (Merrit A J et al. 1994. Cancer Res 54:614-617; Iwadate Yet al. 1996. Int J Cancer 69:236-240; Müllauer L et al. 1996. Hepatology23: 840-847; Newcomb E W. 1995. Leuk Lymphoma 17: 211-221). Theexpression of bcl-2 is considered to be a predictive factor for worseprognosis in prostate and colonic cancer and in neuroblastoma (ThompsonC B. 1995. Science 267: 1456-1462). It has been shown that a number ofantineoplastic therapies induce apoptosis in tumor cells (for reviewssee: Sun S Y et al. 2004. J Natl Cancer Inst. 96:662-672;Schulze-Bergkamen H and Krammer P H. 2004. Semin Oncol. 31:90-119; AbendM. 2003. Int J Radiat Biol. 79:927-941).

Defects in the apoptosis may lead to autoimmune diseases such as lupuserythematosus (Carson DA. and Rebeiro J M. 1993. Lancet. 341: 1251-1254.Aringer M. et al. 1994. Arthritis Rheum. 37:1423-1430), rheumatoidarthritis (Liu H. and Pope R M. 2003. Curr Opin Pharmacol. 3:317-22.)and myasthenia gravis (Masunnaga A. et al. 1994. Immunol Lett. 39:169-172.). Pathogens, such as adenovirus, EBV, cowpox and chlamydia(Thompson C B. 1995. Science 267:1456-1462; Marshall W L. et al. 1999.J. Virol. 73:5181-5185, Deveraux Q L, et al. 1999. J Clin Immunol.19:388-98, Fan T. et al. 1998. J Exp Med. 187:487-496.) have also beenshown to interfere with cellular apoptosis. In chronic inflammatory,hyperproliferative skin diseases such as psoriasis, an abnormally lowrate of apoptosis contributes to the development of epidermalhyperplasia. It was shown that keratinocytes respond to a variety ofexternal and internal growth factors, including some proinflammatorycytokines which may suppress keratinocytes apoptosis, such as IL-15(Ruckert R. et al. 2000. J. Immunol. 165:2240-2250).

Thus, according to another aspect of the present invention, there isprovided a method of enhancing apoptosis of a cell, the methodcomprising providing to the cell an effective concentration of aribonuclease of the T2 family having an actin binding activity, or apolynucleotide encoding and capable of expressing in vivo theribonuclease of the T2 family, thereby enhancing apoptosis. Enhancingapoptosis of a cell by the method of the present invention can beapplied clinically, in the treatment and/or prevention of diseasesassociated with apoptosis, such as the neoplastic diseases, autoimmunediseases, inflammatory disease, hyperproliferative disease andinfectious diseases described hereinabove. Administration of aribonuclease of the T2 family having an actin binding activity, or apolynucleotide encoding and capable of expressing in vivo theribonuclease of the T2 family for enhancing apoptosis in cells of asubject in need thereof can be effected by any of the methods describedherein. Detection of enhanced apoptosis, and the monitoring of changesin the level of apoptosis in cells or tissues or tissue samplesfollowing exposure to T2 RNase, can be effected by cytological,pathology and biochemical (for example, the TUNEL assay describedhereinbelow) means known in the art, as described hereinbelow. Theribonuclease of the T2 family having an actin binding activity can be arecombinant T2 RNase, expressed in a heterologous expression system.Whereas the actin binding and therapeutic character of ribonucleases ofthe T2 family having actin binding activity have been shown to beseparate and independent of the ribonucleolyic activity, in oneembodiment the T2 RNase is devoid of ribonucleolytic activity. Thetherapeutically effective amount of a ribonuclease of the T2 family orof a polynucleotide encoding and capable of expressing in vivo arecombinant ribonuclease of the T2 family can be administered to thecells subject in need thereof, either per se or as an active ingredientof a pharmaceutical composition.

Thus, according to another aspect of the present invention there isprovided a pharmaceutical composition comprising, as an activeingredient, a ribonuclease of the T2 family a polynucleotide encodingand capable of expressing in vivo a recombinant ribonuclease of the T2family, and a pharmaceutically acceptable carrier.

According to yet another aspect of the present invention there isprovided a method of preparing a medicament useful in preventing,inhibiting and/or reversing proliferation, colonization, differentiationand/or development of abnormally proliferating cells comprising the stepof combining a ribonuclease of the T2 family or a polynucleotideencoding and capable of expressing in vivo a recombinant to ribonucleaseof the T2 family, with a pharmaceutically acceptable carrier.

Yet further, there is provided a method of preparing a medicament usefulin treating and/or preventing a disease or condition characterized byexcessive cell motility and/or abnormal accumulation of cells. Themethod is effected by combining a ribonuclease of the T2 family or apolynucleotide encoding and capable of expressing in vivo a recombinantribonuclease of the T2 family, with a pharmaceutically acceptablecarrier.

The medicament is preferably identified as providing a treatment for aspecified proliferative disorder or disease, such as a specified cancer.Such an identification can be made in print on, for example, a containercontaining the medicament or on a leaflet, as is well known in the art.

The method and pharmaceutical composition of the present invention canbe used for, for example, (i) treating a tumor in a subject; (ii)preventing, inhibiting and/or reversing the development a tumor in asubject; (iii) preventing, inhibiting and/or reversing transformation ofa benign tumor to a malignant tumor in a subject; (iv) preventing,inhibiting and/or reversing tumor angiogenesis in a subject; (v)reducing the number of individual tumors in a subject; (vi) reducingtumor size in a subject; (vii) reducing a number of malignant tumors ina subject; and/or (viii) preventing, inhibiting and/or reversingtransformation of a tissue into a tumor in a subject.

The T2-RNase can be derived from a native source, as is furtherexemplified in Example 1 that follows, or alternatively, it can beproduced as a recombinant protein using an appropriate polynucleotide(see Table 3 below and the following descriptions) and expressionsystem. Expressing and purifying recombinant proteins is well known inthe art and can be effected by any one of a plurality of alternativetechniques described in detail in any one of a number of text books andlaboratory protocol books, including, for example, “Molecular Cloning: Alaboratory Manual” Sambrook et al., (1989); “Current Protocols inMolecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel etal., “Current Protocols in Molecular Biology”, John Wiley and Sons,Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”,John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”,Scientific American Books, New York; Birren et al. (eds) to “GenomeAnalysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring HarborLaboratory Press, New York (1998).

TABLE 3 GeneBank Accession Source Name(Prot) Name(Gene) Reference(s) No.Bacteria Aeromonas Locus RNI Favre, D. et al. 1993. Q07465 hydrophila J.Bacteriol. 175: 3710-3722. Haemophilus Rnase Locus RN26 Fleischmann, R.D., et al. 1995. P44012 influenzae HI0526 Science 269: 496-512.Escherichia coli Rnase I Locus RNI Meador, J. III. & Kennell, D. 1990.P21338 Gene 95: 1-7. Oshima, T., et al. 1996. DNA Res. 3: 137-155.Henikoff, S. & Henikoff, J. G. 1994. Genomics 19: 97-107. Aspergillusoryzae Rnase T2 rnt B Kawata Y. et al. 1988. P10281 Eur J. Biochem176(3): 683-97. Kawata Y. et al. 1990. Eur J. Biochem 187: 255-62. OzekiK, et al. 1991. Curr Genet. 19: 367-73. Fungi Rhisopus niveus RnaseHoriuchi, H. et al. 1988. P08056 Rh J. Biochem. 103: 408-418. Kurihara,H. et al. 1992. FEBS Lett. 306: 189-192. Kurihara, H. et al. 1996. J.Mol. Biol. 255: 310-320. Ohgi, K. et al. 1991. J. Biochem. 109: 776-785.Trichoderma viride Rnase Inada, Y. et al. 1991. J. P24657 Trv Biochem.110 (6), 896-904. Lentinula edodes Rnase Kobayashi, H. et al. 1992.AAB24971 (shiitake mushroom) Irp Biosci. Biotechnol. Biochem. 56:2003-2010. L. edodes Rnase Kobayashi, H. et al. 1992. P81296 Le2 Biosci.Biotechnol. Biochem. 56: 2003-2010. Shimada, H. et al. 1991. Agric.Biol. Chem. 55: 1167-1169. Irpex lacteus Rnase Watanabe, H., et al.1995. AAB35880 Irp1 Biosci. Biotechnol. Biochem. 59: 2097-2103. PhysarumRnase Inokuchi, N. et al. 1993. P81477 polycephlum Phyb J. Biochem. 113:425-432. Plants Arabidopsis thaliana RNS2 Locus Green, P. J. 1993.P42814 RNS2 Proc. Natl. Acad. Sci. U.S.A. 90: 5118-5122. A. thalianaRnase 3 Locus Bariola, P. A., et al. 1994. P42815 RNS3 Plant J. 6:673-685. A. thaliana Rnase 1 Locus Bariola, P. A., et al. 1994. P42813RNS1 Plant J. 6: 673-685. Lycopersicon Rnase LE RNALE Kock, M. et al.1995. P80022 esculentum Plant Mol. Biol. 27: 477-485. (cultured tomato)Jost, W. et al. 1991. Eur. J. Biochem. 198: 1-6. L. esculentum RnaseRNLX Kock, M., et al. 1995. P80196 LX Plant Mol. Biol. 27: 477-485.Loffler, A., et al. 1993. Eur. J. Biochem. 214: 627-633. Nicotiana alataS-RNase S Anderson, M. A., et al. 1986. P04002 (tobacco) Nature 321:38-44. Matton, D. P. et al. 1995. Plant Mol. Biol. 28: 847-858. McClure,B. A. et al. 1989. Nature 342: 95-97. Malus domestica S-RNases S Sassa,H., et al. 1996. (apple tree) Mol. Gen. Genet. 250: 547-557. Pyruspyrifolia S-RNases S Norioka, N., et al. 1996. (Japanese pear) J.Biochem. 120; 335-345. Momordica charantia RNase Locus Blaxter, M. L.,et al. 1996. P23540 (bitter gourd) MC RNMC Mol. Biochem. Parasitol. 77:77-93. Ide, H. et al. 1991. FEBS Lett. 284: 161-164. Ide, H. et al.1991. FEBS Lett. 289: 126. Animals Gallus gallus RNase Uchida, T. et al.1996. JC5126 (chicken) CL1 Biosci. Biotechnol. Biochem. 60: 1982-1988.Rana catesbeiana RNase Yagi, H. et al. 1995. PC2347 (bull frog) RCL2Biol. Pharm. Bull. 18: 219-222. Liao, Y. D. et al. 1996. Protein ExprPurif. 7: 194-202. Liao Y D, et al. 1994. Eur J Biochem. 222: 215-20.Liao, Y. D. et al. 1998. J. Biol. Chem. 273: 6395-401 Drosophyla RNaseDmRNase Lankenau, D. H. et al. 1990. X15066 melanogaster DM Chromosoma99: 111-117. Hime, G., et al. 1995. Gene 158: 203-207. Crassostera gigusRNase Locus Watanabe, H. et al. 1993. JX029 (pacific oyster.) Oy JX0295J. Biochem. 114: 800-807. Todarodes pasificus RNase Kusano, A. et al.1998. PMID (Japanese Tp Biosci. Biotechnol. Biochem. 9501521 flyingsquid) 62: 87-94. Homo sapiens RNase RNase6PL Trubia, M. et al. 1997.NP003721 6 precurs. Genomics 42: 342-344.

For some applications it may be beneficial to use a ribonuclease whichsubstantially lacks ribonucleolytic activity, which may have or causeundesired side effects. As used herein the phrase “substantially lacksribonucleolytic activity” refers to (i) an inactivated ribonuclease(either natural or recombinant) of the T2 family which has 0-10%ribonucleolytic activity as is compared to a similar, non-inactivated,ribonuclease; and/or (ii) a recombinant mutant (natural isolate or maninduced) ribonuclease of the T2 family which has 0-10% ribonucleolyticactivity as is compared to a similar, non-mutant, ribonuclease.Inactivating the ribonucleolytic to activity of the ribonuclease of theT2 family may be effected by a process selected from the groupconsisting of, autoclaving and chemically denaturing or inactivating.

As used herein, the term “autoclaving” is defined as exposure, in anautoclave, to superheated steam heated to about 121° C. at high pressureconditions (for example, 15 psi), for at least 20 minutes. Chemicaldenaturing may be effected by exposure to extremes of pH, and chemicalagents causing alteration in the amino acid side chains and/or chemicalbond structure of the ribonuclease of the T2 family, such asiodoacetylation, as described in detail hereinbelow (Example 6).

As is further detailed in Examples 2 and 6 below, it has been shown bythe inventors of the present invention that the inhibition of cellmotility, the inhibition of apoptosis, the anti-proliferation,anti-colonization, anti-differentiation and/or anti-developmentactivities of RNase B1 are not dependent on its ribonucleolyticactivity, as boiled, autoclaved and chemically inactivated (acetylated)RNase B1, which has little (10%) or substantially no (0-10%)ribonucleolytic activity retained substantially all of its actin-bindingand anti-proliferation, anti-colonization, anti-differentiation and/oranti-development activities.

Thus, a T2-RNase protein according to the present invention can beutilized both in a native, ribonucleolytic active, form, or,alternatively, in a silent, or repressed ribonucleolytic form, having no(0%) or little (up to 10%) ribonucleolytic activity, yet which retainsits other activities. As such, the term “T2-RNases” is meant toencompass all the anti-proliferation, anti-colonization,anti-differentiation and/or anti-development forms of the protein,regardless of other activities thereof. Thus, in one embodiment, theT2-RNase of the present invention is substantially devoid ofribonuclease activity yet has an actin binding activity. In a preferredembodiment which actin binding activity of the T2 RNase is thermostable.Further, there is to provided a method inactivating a ribonucleaseactivity, yet maintaining an actin binding activity of a ribonuclease ofthe T2 family. The method is affected by subjecting the ribonuclease todenaturing conditions sufficient for substantially inactivating theribonuclease activity, yet maintaining the actin binding activity.

It will be appreciated that utilizing a T2-RNase, either directly orexpressed from a polynucleotide, which displays a desired activity andyet is devoid of, or repressed in, ribonucleolytic activity isparticularly advantageous since ribonucleolytic activity can produceundesired side effects in a subject.

A polypeptide representing the amino acid sequence of a T2-RNase asdefined herein can be produced by any one of several methods well knownin the art. For example the polypeptide can be produced synthetically bystandard peptide synthesis techniques, for example using either standard9-fluorenylmethoxycarbonyl (F-Moc) chemistry (see, for example,Atherton, E. and Sheppard, R. C. 1985, J. Chem. Soc. Chem. Comm 165) orstandard butyloxycarbonate (T-Boc) chemistry, although it is noted that,more recently, the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl system,developed by Sheppard has found increasingly wide application (Sheppard,R. C. 1986 Science Tools, The LKB Journal 33, 9).

While reducing the present invention to practice, it was uncovered, forthe first time, that many ribonucleases of the T2 family, derived fromorganisms of diverse phylogentic origin from bacteria to yeast (Example16, FIG. 47 a-47 b) have actin binding activity, which correlates withthe therapeutic character and inhibition of cell motility, abnormal cellproliferation and apoptosis. Thus, the T2-RNase protein can also beisolated and purified by methods well known in the art from organismsknown to express this protein. Such organisms include, for example,Aeromonas hydrophila, Haemophilus influenzae, Escherichia coli,Aspergillus oryzae, Aspergillus phoenicis, Rhisopus niveus, Trichodermaviride, Lentinula edodes, Irpex lacteus; Physarum polycephlum,Arabidopsis thaliana, Lycopersicon esculentum, Nicotiana alata, Malusdomestica, Pyrus pyrifolia, Momordica charantia, Gallus gallus, Ranacatesbeiana, Drosophyla melanogaster, Crassostera gigus, Todarodespasificus and Homo sapiens. It is, however, anticipated that otherorganisms yet not known to produce RNase, once uncovered as such, couldalso be used as a source for T2-RNase according to the presentinvention.

It will be appreciated that some therapeutic and diagnostic use of T2ribonucleases of the present invention will require purified T2 RNases.Thus, simple, inexpensive methods of purification of T2 RNases areadvantageous. While reducing the present invention to practice, it wasuncovered that recombinant ribonuclease of the T2 family can be isolatedand purified by boiling, fractionation by column chromatography (seeExample 19, FIG. 54 hereinbelow), and assaying the collected fractionsfor ribonucleolytic activity. Thus, there is also provided a novelmethod for isolating T2 ribonuclease protein, the method comprising heatdenaturating a T2 containing sample which comprises cells expressing aT2 ribonuclease protein, separating the supernatant, preferably bycentrifigation, fractionating the supernatant, identifying a fraction ofthe supernatant having a T2 ribonuclease protein and purifying the T2RNase fraction to substantial purity. In one embodiment, fractionatingthe supernatant is effected by column chromatography, for example, QSEPHAROSE or other protein separation media well known to one ofordinary skill in the art. Identification of the T2 RNase bearingfractions may be made according to physical characteristics (such aselectrophoretic mobility) and functional criteria (ribonucleolyticactivity, actin binding activity). Methods for physical and functionalassessment of the purified RNase are well known in the art, as describedhereinbelow.

Alternatively and preferably a T2-RNase protein can be recombinantlyproduced by expressing a polynucleotide encoding same, using anappropriate expression vector system. In one embodiment, the expressionsystem is a heterologous expression system selected from a bacterial,yeast, or higher cell expression system, wherein higher cell expressionsystems include animal or plant expression systems. Preferably, anexpression system is selected which provides suitable post translationalmodifications. Suitable expression vector systems include, but are notlimited to, mammalian cells infected with a virus (e.g., adenovirus,retrovirus, herpes simplex virus, avipox virus); insect cells infectedwith a virus (e.g., baculovirus); genetically modified plants or plantcells transformed with a plasmid, a plant virus or an Agrobacterium;transformed microorganisms such as yeasts containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.The expression controlling elements of vectors vary in their strengthsand specifications depending on the host-vector system utilized, any oneof a number of to suitable transcription and translation elements may beused. A recombinantly produced T2-RNase may be purified from host cellsby the methods described hereinbelow, or by affinity chromatography,electrophoresis, high-performance liquid chromatography (HPLC),immunoprecipitation, sedimentation or any other method known to the art.

A purified T2-RNase can be used to prepare a medicament according to thepresent invention by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or lyophilizing processes with the addition of theappropriate pharmaceutically acceptable carriers and/or excipients oralternatively it can be linked to appropriate delivery vehicles asdescribed hereinabove.

A polynucleotide according to the present invention can encode a nativeT2-RNase protein, which term in this context to describe a T2-RNasehaving both anti-proliferative and ribonucleolytic activities, oralternatively, a polynucleotide according to the present invention canencode a silent or repressed T2-RNase mutant, having no or littleribonucleolytic activity, to be expressed (e.g., transcribed andtranslated) in vivo into a protein which is substantially free ofribonucleolytic activity.

As such, the term “polynucleotide” when used herein in context ofT2-RNases in general, or in context of any specific T2-RNase, refers toany polynucleotide sequence which encodes a T2-RNase active inpreventing, inhibiting and/or reversing proliferation, colonization,differentiation and/or development of abnormally proliferating cells,either having or substantially devoid of ribonucleolytic activity.Polynucleotides encoding a T2-RNase devoid of ribonucleolytic activitycan be obtained using known molecular biology techniques, such as randommutagenesis, site-directed mutagenesis and enhanced evolutiontechniques. Site directed mutagenesis can be readily employed becausethe amino acid residues essential for the ribonucleolytic activity ofT2-RNases have been recognized (see Kusano et al., 1998. Biosci.Biothechnol. Biochem. 62:87-94, which is incorporated herein byreference, and Table 3 above).

It will be appreciated that aberrant or excessive or insufficientregulation of actin function and intracellular actin distribution andcellular motility can lead to perturbed cellular functions, which inturn can lead to cellular disorders (or exacerbate to existing cellulardisorders). As used herein, a “cellular disorder” includes a disorder,disease, or condition characterized by aberrant or insufficient cellularability to move or migrate properly in response to certain stimuli (e.g.tissue damage), or inability to properly regulate actin function anddistribution within the cell.

While reducing the present invention to practice, it was shown, for thefirst time, that T2 RNase not only inhibits cell motility, actinfilament assembly and reassembly, tube formation and cel proliferationin vivo, but can also directly inhibit the motility and invasiveness ofcancer cells, as measured in vitro (Example 12, Table 5). Thus, the T2RNases having actin binding activity of the present invention, andcompositions comprising such, may act as novel therapeutic agents forcontrolling cellular disorders related to motility, including cancer(e.g. tumor angiogenesis and metastasis), immune regulation,neurodegenerative and inflammatory disease. Additionally, the T2 RNaseshaving actin binding activity may act as novel therapeutic agents forameliorating certain cellular disorders and conditions through theirability to migrate and to regulate tissue injury response.

Thus, according to the present invention there is provided a method ofinhibiting motility of a cell. The method is effected by providing tothe cell a ribonuclease of the T2 family having an actin bindingactivity, or a polynucleotide encoding and capable of expressing in vivothe ribonuclease of the T2 family. Further there is provided a method oftreating and/or preventing a disease or condition characterized byexcessive cell motility in a subject in need thereof. The method iseffected by administering to the subject a therapeutically effectiveamount of a ribonuclease of the T2 family having an actin bindingactivity, or a polynucleotide encoding and capable of expressing in vivosaid ribonuclease of the T2 family.

Disruption of actin assembly and disassembly affects cell motility,development, growth, proliferation and reproduction. Thus, thecompositions and methods of present invention can be used for treatingconditions, syndromes or diseases characterized by abnormal accumulationof cells. Diseases or conditions characterized by abnormal accumulationof cells include, but are not limited to, inflammatory diseases,neurodegenerative diseases, and cancer. Further, the compositions andmethods of the present invention can be used for inhibiting actin tofilament assembly and disassembly in a cell, effected by providing tothe cell a ribonuclease of the T2 family having actin binding activity,or a polynucleotide encoding and capable of expressing in-vivo theribonuclease of the T2 family.

Thus, the present invention can be used for treating conditions,syndromes or diseases characterized by abnormally proliferating cells,such as cancerous or other cells, such as, but not limited to, amalignant or non-malignant cancer including biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; endometrialcancer; esophageal cancer; gastric cancer; intraepithelial neoplasms;lymphomas; lung cancer (e.g. small cell and non-small cell); melanoma;neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostatecancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroidcancer; and renal cancer, as well as other carcinomas and sarcomas,papilloma, blastoglioma, Kaposi's sarcoma, squamous cell carcinoma,astrocytoma, head cancer, neck cancer, bladder cancer, colorectalcancer, thyroid cancer, pancreatic cancer, gastric cancer,hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease,Burkitt's disease, arthritis, rheumatoid arthritis, diabeticretinopathy, angiogenesis, restenosis, in-stent restenosis, vasculargraft restenosis, proliferative vitreoretinopathy, chronic inflammatoryproliferative disease, dermatofibroma and psoriasis.

As used herein the terms “cancer” or “tumor” are clinically descriptiveterms which encompass a myriad of diseases characterized by cells thatexhibit abnormal cellular proliferation. The term “tumor”, when appliedto tissue, generally refers to any abnormal tissue growth, characterizedin excessive and abnormal cellular proliferation. A tumor may be“benign” and unable to spread from its original focus, or “malignant” or“metastatic” and capable of spreading beyond its anatomical site toother areas throughout the host body. The term “cancer” is an older termwhich is generally used to describe a malignant tumor or the diseasestate arising therefrom. Alternatively, the art refers to an abnormalgrowth as a neoplasm, and to a malignant abnormal growth as a malignantneoplasm.

The T2 RNase having an actin binding activity of the present inventioncan be used in the preventive treatment of a subject at risk of having acancer. A “subject at risk of having a cancer” as used herein is asubject who has a high probability of developing cancer. These subjectsinclude, for instance, subjects having a genetic to abnormality, thepresence of which has been demonstrated to have a correlative relationto a higher likelihood of developing a cancer and subjects exposed tocancer causing agents such as tobacco, asbestos, or other chemicaltoxins, or a subject who has previously been treated for cancer and isin apparent remission. When a subject at risk of developing a cancer isexposed to the T2 RNase having actin binding activity of the presentinvention, the subject may be able to prevent any cancer that does formfrom becoming metastatic.

The T2 RNase having an actin binding activity of the present inventionis also useful for treating and/or preventing disorders associated withinflammation in a subject. Immune or hematopoietic cells exposed to T2RNases having an actin binding activity would have a reduced ability tomigrate. Thus T2 RNases having actin binding activity is useful forpreventing inflammation associated with immune cell migration and fortreating and preventing inflammatory disorders and ischemic diseases.

Inflammatory disorders and ischemic diseases are characterized byinflammation associated with neutrophil migration to local tissueregions that have been damaged or have otherwise induced neutrophilmigration and activation. While not intending to be bound by anyparticular theory, it is believed that excessive accumulation ofneutrophils resulting from neutrophil migration to the site of injury,causes the release toxic factors that damage surrounding tissue. Whenthe inflammatory disease is an acute stroke a tissue which is oftendamaged by neutrophil stimulation is the brain. As the activeneutrophils accumulate in the brain an infarct develops.

An “inflammatory disease or condition” as used herein refers to anycondition characterized by local inflammation at a site of injury orinfection and includes autoimmune diseases, certain forms of infectiousinflammatory states, undesirable neutrophil activity characteristic oforgan transplants or other implants and virtually any other conditioncharacterized by unwanted neutrophil accumulation at a local tissuesite. These conditions include but are not limited to meningitis,cerebral edema, arthritis, nephritis, adult respiratory distresssyndrome, pancreatitis, myositis, neuritis, connective tissue diseases,phlebitis, arteritis, vasculitis, allergy, anaphylaxis, ehrlichiosis,gout, organ transplants and/or ulcerative colitis.

An “ischemic disease or condition” as used herein refers to a conditioncharacterized by local inflammation resulting from an interruption inthe blood supply to a tissue due to a blockage or hemorrhage of theblood vessel responsible for supplying blood to the tissue such as isseen for myocardial or cerebral infarction. A cerebral ischemic attackor cerebral ischemia is a form of ischemic condition in which the bloodsupply to the brain is blocked. This interruption in the blood supply tothe brain may result from a variety of causes, including an intrinsicblockage or occlusion of the blood vessel itself, a remotely originatedsource of occlusion, decreased perfusion pressure or increased bloodviscosity resulting in inadequate cerebral blood flow, or a rupturedblood vessel in the subarachnoid space or intracerebral tissue.

In some aspects of the invention the T2 RNase of the present inventionis provided in an effective amount to prevent migration of a tumor cellacross a barrier. The invasion and metastasis of cancer is a complexprocess which involves changes in cell adhesion properties which allow atransformed cell to invade and migrate through the extracellular matrix(ECM) and acquire anchorage-independent growth properties. Liotta, L.A., et al., Cell 64:327-336 (1991). Some of these changes occur at focaladhesions, which are cell/ECM contact points containingmembrane-associated, cytoskeletal, and intracellular signalingmolecules. Metastatic disease occurs when the disseminated foci of tumorcells seed a tissue which supports their growth and propagation, andthis secondary spread of tumor cells is responsible for the morbidityand mortality associated with the majority of cancers. Thus the term“metastasis” as used herein refers to the invasion and migration oftumor cells away from the primary tumor site.

The T2 RNase or polynucleotide encoding same of the present inventioncan be used to assay cells for sensitivity to inhibition of cellularmotility, for example, in testing their ability to cross a barrier.Preferably the tumor cells are prevented from crossing a barrier. Thebarrier for the tumor cells may be an artificial barrier in vitro or anatural barrier in vivo. In vitro barriers include but are not limitedto extracellular matrix coated membranes, such as Matrigel. Thus, T2RNase can be provided to cells which can then be tested for theirability to inhibit tumor cell invasion in a Matrigel invasion assaysystem as described in detail by Parish, C. R., et al., “ABasement-Membrane Permeability Assay which Correlates with theMetastatic Potential of Tumour Cells,” Int. J. Cancer (1992) 52:378-383.Matrigel is a reconstituted basement membrane containing type IVcollagen, laminin, heparan sulfate proteoglycans such as perlecan, whichbind to and localize bFGF, vitronectin as well as transforming growthfactor-.beta. (TGF-.beta.), urokinase-type plasminogen activator (uPA),tissue plasminogen activator (tPA), and the serpin known as plasminogenactivator inhibitor type 1 (PAI-1). Other in vitro and in vivo assaysfor metastasis have been described in the prior art, see, e.g., U.S.Pat. No. 5,935,850, issued on Aug. 10, 1999, which is incorporatedherein by reference. An in vivo barrier refers to a cellular barrierpresent in the body of a subject.

Any ribonuclease of the T2 family which has the actin binding,anti-proliferation, anti-colonization, anti-differentiation and/oranti-development activities described herein and exemplified in theExamples section that follows can be used as a therapeutic agent inaccordance with the teachings of the present invention. Similarly, anypolynucleotide encoding a ribonuclease of the T2 family which has theanti-proliferation, anti-colonization, anti-differentiation and/oranti-development activities described herein can be used as atherapeutic agent in accordance with the teachings of the presentinvention. A non exhausting list of ribonucleases of the T2 family isprovided in Table 3, above. As is further exemplified by the Examplesthat follow, RNase B1, which is a member of the T2 family, hasanti-proliferation, anti-colonization, anti-differentiation and/oranti-development activities as was determined by in vivo and in vitroassays. In addition, RNase B1 is shown to bind to actin even whentreated so as to render it free of ribonuclease activity. Thus, thepresent invention provides three different assays with which one ofordinary skills in the art could test a given ribonuclease for its actinbinding, anti-proliferation, anti-colonization, anti-differentiationand/or anti-development activities, these are an in vitro assay fordetermining the effect of the tested ribonuclease on cancerous cells, invivo assay for determining the effect of the tested ribonuclease ontumor development, and another in vitro assay for determining theability of the tested ribonuclease to bind to cellular and/or freeactin. Without limiting the present invention by any theory, it isbelieved that an ability of a ribonuclease to bind to actin isindicative that such a ribonuclease has anti-proliferation,anti-colonization, anti-differentiation and/or anti-developmentactivities.

A ribonuclease according to the present invention can be administered toan organism, such as a human being or any other mammal, per se, or in apharmaceutical composition where it is mixed with suitable carriers orexcipients.

As used herein a “pharmaceutical composition” or “medicament” refers toa preparation of one or more of the ribonucleases or polynucleotidesencoding same as described herein, with other chemical components suchas physiologically suitable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of a compoundto an organism.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Pharmaceutical compositions may also include one or more additionalactive ingredients, such as, but not limited to, anti inflammatoryagents, antimicrobial agents, anesthetics, cancer therapeutic agents andthe like in addition to the main active ingredient. A detaileddescription of commonly used additional agents suitable for use with thecompositions of the present invention is presented hereinbelow.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

While reducing the present invention to practice, significanttherapeutic effects of the ribonuclease of the T2 family having an actinbinding activity were revealed using a broad variety of means ofadministration, in diverse models of abnormal cell proliferation andaccumulation. Intraperitoneal administration, providing rapid systemicuptake and distribution of the T2 RNase, was found effective insuppressing tumor growth and development in subcutaneous tumors in nudemice (Examples 9 and 13, FIGS. 35 and 39 a-b) and intraperitoneal tumors(Example 8, FIGS. 33 a-d). Intravenous administration, providing evenmore rapid systemic uptake of the T2 RNase, was also found effective insuppressing and treating subcutaneous xenografts (Example 13, FIGS. 36a-b), and remote (lung) metastatic spread of intravenous tumors (Example8, FIGS. 34 a-e). Direct administration of, and preincubation of cellswith the T2 RNase has been found effective in preventing tumor growth inbreast carcinoma (Example 12, Table 6), colon carcinoma (Example 12,Table 6), melanoma (Example 12, FIG. 38), in-vivo, angiogenic factorinduced angiogenesis and microvessel density (Example 10, FIG. 37) andcell tube formation in both plant (Example 1, FIGS. 8 and 9) and humanHUVE cells (Example 7, FIGS. 32 a-h). Oral administration of T2 RNase,in the form of microcapsules, has been found effective in reducing tumorproliferation, tumor size, tumor vascularization and the number ofaberrant crypt foci (Example 4, FIGS. 23 a-23 c, 24 a-24 d, and 25 a-25c) when administered early in colon tumor (DMH model) induction. Similaroral administration of T2 RNase to animals harboring already welldeveloped tumors reduced the degree of vascularization and malignancy ofcolon cancer tumors in rats (Example 4, FIG. 27 c), despite exposure ofthe RNase to digestive processes and low doses presumed deliveredintraintestinally. It will be appreciated that encapsulation methodsproviding effective intestinal release of compositions are well known inthe art, and use of such is expected to increase the effectiveness oforal administration of T2 RNase in cases of already established tumors.

Thus, to effect administration the pharmaceutical composition of thepresent invention includes a suitable pharmaceutical carrier and aneffective amount of a T2-RNase or a polynucleotide encoding same, and isadministered, for example, topically, intraocularly, parenterally,orally, intranasally, intravenously, intramuscularly, subcutaneously orby any other effective means via methods well known in the art.

For intravenously, intramuscularly or subcutaneously injection, aT2-RNase or a polynucleotide encoding same may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHank's solution, Ringer's solution, or physiological saline buffer. Forexample, a physiologically appropriate solution to containing aneffective amount of a T2-RNase or a polynucleotide encoding same can beadministered systemically into the blood circulation to treat a canceror tumor which cannot be directly reached or anatomically isolated. Aphysiologically appropriate solution containing an effective amount of aT2-RNase or a polynucleotide encoding same may be directly injected intoa target cancer or tumor tissue by a needle in amounts effective totreat the tumor cells of the target tissue.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the pharmaceutical composition of the presentinvention can be formulated readily by combining a T2-RNase or apolynucleotide encoding same with pharmaceutically acceptable carrierswell known in the art. Such carriers enable a T2-RNase or apolynucleotide encoding same to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for oral ingestion by a patient. Pharmacological preparations fororal use can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Additional pharmaceutical compositions, which can be used orally,include to push-fit capsules made of gelatin as well as soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules may contain a T2-RNase or apolynucleotide encoding same in admixture with filler such as lactose,binders such as starches, lubricants such as talc or magnesium stearateand, optionally, stabilizers. In soft capsules, a T2-RNase or apolynucleotide encoding same may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for the chosen routeof administration.

Oral delivery of the pharmaceutical composition of the present inventionmay not be successful due to the pH and enzyme degradation present inthe gastrointestinal tract. Thus, such pharmaceutical compositions mustbe formulated to avoid undesirable circumstances. For example, entericcoating can be applied to oral solid formulation. Substances withacidic-resistant properties such as cellulose acetate phtalate (CAP),hydroxypropyl methycellulose phtalate (HPMCP) and acrylic resins aremost commonly used for coating tablets or granules for microencapsulation. Preferably wet granulation is used to prepare theenteric-coated granules to avoid reactions between the active ingredientand the coating (Lin, S. Y. and Kawashima, Y. 1987, Pharmaceutical Res.4:70-74). A solvent evaporation method can also be used. The solventevaporation method was used to encapsulate insulin administered todiabetic rats to maintain blood glucose concentration (Lin, S. Y. etal., 1986, Biomater, Medicine Device, Artificial organ 13:187-201 andLin, S. Y. et al., 1988, Biochemical Artificial Cells Artificial Organ16:815-828). It was also used to encapsulate biological materials ofhigh molecular weight such as vial antigen and concanavalin A (Maharaj,I. Et al. 1984, J. Phamac. Sci. 73:39-42).

For buccal administration, the pharmaceutical composition of the presentinvention may take the form of tablets or lozenges formulated inconventional manner.

For rectal administration propositories can be used as is well known inthe art.

For administration by inhalation, a T2-RNase or a polynucleotideencoding same for use according to the present invention is convenientlydelivered in the form of an aerosol spray presentation from apressurized pack or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of a T2-RNase or a polynucleotide encoding sameand a suitable powder base such as lactose or starch.

The pharmaceutical composition of the present invention may also beformulated for parenteral administration, e.g., by bolus injection orcontinuos infusion. A composition for injection may be presented in unitdosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. The compositions may be suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of a T2-RNase or a polynucleotide encodingsame may be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofa T2-RNase or a polynucleotide encoding same to allow for thepreparation of highly concentrated solutions.

Alternatively, a T2-RNase or a polynucleotide encoding same may be in apowder form for constitution with a suitable vehicle, e.g., sterile,pyrogen-free water, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In addition, a cancer or tumor present in a body cavity, such as in theeye, gastrointestinal tract, genitourinary tract (e.g., the urinarybladder), pulmonary and bronchial system and the like, can receive aphysiologically appropriate composition (e.g., a solution such as asaline or phosphate buffer, a suspension, or an emulsion, which issterile) containing an effective amount of a T2-RNase or apolynucleotide encoding same via direct injection with a needle or via acatheter or other delivery tube placed into the cancer or tumorafflicted hollow organ. Any effective imaging device such as X-ray,sonogram, or fiber optic visualization system may be used to locate thetarget tissue and guide the needle or catheter tube in proximitythereto.

The pharmaceutical composition of the present invention can also bedelivered by osmotic micro pumps. The osmotic micro pumps are implantedinto one of the body cavities and the drug is constantly released ontothe tissue to be treated. This method is particularly advantageous whenan immune response to the pharmaceutical composition is experienced.This method has been employed for ONCONASE (Vasandani V. M., et al.,1996, Cancer Res. 15; 56(18):4180-6).

Alternatively and according to another preferred embodiment of thepresent invention, the pharmaceutically acceptable carrier includes adelivery vehicle capable of delivering a T2-RNase or a polynucleotideencoding same to the mammalian cell of the subject.

Numerous delivery vehicles and methods are known in the art fortargeting proteins or nucleic acids into or onto tumors or cancer cells.For example, liposomes are artificial membrane vesicles that areavailable to deliver proteins or nucleic acids into target cells(Newton, A. C. and Huestis, W. H., Biochemistry, 1988, 27:4655-4659;Tanswell, A. K. et al., 1990, Biochmica et Biophysica Acta,1044:269-274; and Ceccoll, J. et al., Journal of InvestigativeDermatology, 1989, 93:190-194). Thus, a T2-RNase or a polynucleotideencoding same can be encapsulated at high efficiency with liposomevesicles and delivered into mammalian cells. In addition, the T2-RNaseprotein or nucleic acid can also be delivered to target tumor or cancercells via micelles as described in, for example, U.S. Pat. No. 5,925,628to Lee, which is incorporated herein by reference.

Liposome or micelle encapsulated T2-RNase or a polynucleotide encodingsame may be administered topically, intraocularly, parenterally,intranasally, intratracheally, intrabronchially, intramuscularly,subcutaneously or by any other effective means at a dose efficacious totreat the abnormally proliferating cells of the target tissue. Theliposomes may be administered in any physiologically appropriatecomposition containing an effective amount of encapsulated T2-RNase or apolynucleotide encoding same.

Alternatively and according to another preferred embodiment of thepresent invention the delivery vehicle can be, but it is not limited to,an antibody or a ligand capable of binding a specific cell surfacereceptor or marker. An antibody or ligand can be directly linked to aT2-RNase protein or nucleic acid via a suitable linker, or alternativelysuch an antibody or ligand can be provided on the surface of a liposomeencapsulating a T2-RNase or a polynucleotide encoding same.

For example, a T2-RNase or a polynucleotide encoding same can be fusedwith specific membranal protein antibodies or ligands for targeting tospecific tissues or cells as previously described in the art. It will beappreciated in this respect that fusion of RNase A of the ribonuclease Asuperfamily with antibodies to the transferrin receptor or to the T cellantigen CD5 lead to inhibition of protein synthesis in tumor cellscarrying a specific receptor for each of the above toxins (Rybak, M. etal., 1991, J. Biol. Chem. 266:21202-21207 and Newton D L, et al., 1997,Protein Eng. 10(4):463-70).

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount of theactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the IC₅₀ and theLD₅₀ (lethal dose causing death in 50% of the tested animals) for asubject active ingredient. The data obtained from these cell cultureassays and animal studies can be used in formulating a range of dosagefor use in human. The dosage may vary depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g.,Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p. 1).

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

While reducing the present invention to practice, it was surprisinglyuncovered that administration of the T2 RNase having an actin bindingactivity has a synergistic effect on the anti-tumor efficacy of theTAXOL treatment (Example 15, FIG. 46). Thus, the T2 RNase having anactin binding activity or polynucleotide encoding same of the presentinvention can be used to treat diseases or conditions associated withaberrant cellular motility alone or in combination with otherestablished or experimental therapeutic regimen for such disorders.Thus, according to the present invention there are provided methods ofenhancing therapeutic treatment of a cancer. The methods are effected byadministering to a subject in need thereof, in combination with thetherapeutic treatment, a ribonuclease of the T2 family having an actinbinding activity, or a polynucleotide encoding and capable of expressingin vivo the ribonuclease of the T2 family. It will be appreciated thatsuch synergistic activity of T2 RNase treatment with additionaltherapeutic methods or compositions has the potential to significantlyreduce the effective clinical doses of such treatments, thereby reducingthe often devastating negative side effects and high cost of thetreatment.

Therapeutic regimen for treatment of cancer suitable for combinationwith the T2 RNase of the present invention or polynucleotide encodingsame include, but are not limited to chemotherapy, radiotherapy,phototherapy and photodynamic therapy, surgery, nutritional therapy,ablative therapy, combined radiotherapy and chemotherapy,brachiotherapy, proton beam therapy, immunotherapy, cellular therapy toand photon beam radiosurgical therapy.

Anti-cancer drugs that can be co-administered with the compounds of theinvention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Fluorocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride. Additional antineoplastic agents include those disclosedin Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A.Chabner), and the introduction thereto, 1202-1263, of Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition,1990, McGraw-Hill, Inc. (Health Professions Division).

Anti-inflammatory drugs that can be administered in combination with theT2 RNase having an actin binding activity or polynucleotide encodingsame of the present invention include but are not limited to Alclofenac;Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase;Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride;Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium;Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; toFluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen;Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide;Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen;Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin;Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; IsoflupredoneAcetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride;Lomoxicam; Loteprednol Etabonate; Meclofenamate Sodium; MeclofenamicAcid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium.

As has already been mentioned hereinabove, according to an aspect of thepresent invention, the active ingredient of the pharmaceuticalcomposition is a polynucleotide encoding a T2-RNase.

According to this aspect of the present invention the polynucleotide isintroduced into the mammalian cell along with a pharmaceuticallyacceptable carrier, which introduction results in a genetic modificationof this cell, enabling the expression of a T2-RNase therein.

As used herein in the specification and in the claims section below, theterm “genetic modification” refers to a process of inserting nucleicacids into cells. The insertion may, for example, be effected by viralinfection, injection, transfection, particle bombardment or any othermeans effective in introducing nucleic acids into cells, some of whichare further detailed hereinbelow. Following the genetic modification thenucleic acid is either integrated in all or part, to the cell's genome(DNA), or remains external to the cell's genome, thereby providingstably modified or to transiently modified cells.

As such, the pharmaceutical composition according to this aspect of thepresent invention is usable for gene therapy.

As used herein the phrases “gene therapy” or “genetic therapy” are usedinterchangeably and refer to a method of therapy in which a stable ortransient genetic modification of a proliferative cell(s) such as acancer cell, leads to the inhibition of proliferation of this cell.

Any one of the polynucleotides identified in Table 3 by its Gene Bankaccession number can be employed according to the present invention as apolynucleotide encoding a T2-RNase. In addition, polynucleotides 40% ormore homologous and/or hybridizing under mild and/or stringenthybridization conditions with the listed polynucleotides can also beemployed as a polynucleotide encoding a T2-RNase, provided that theprotein encoded thereby is characterized as a T2-RNase and exhibits thedesired activities. Furthermore, it will be appreciated that portions,mutants chimeras or alleles of such polynucleotides can also be employedas a polynucleotide encoding a T2-RNase according to the presentinvention, again, provided that such portions, mutants chimeras oralleles of such polynucleotides encode a T2-RNase which exhibits thedesired activities.

Isolation of novel polynucleotides encoding T2-RNases is also envisaged.Such isolation can be effected using methodologies well known in the artsuch as, but not limited to, library screening, hybridization, PCRamplification, labeled primers, labeled degenerated primers. Bothgenomic and cDNA polynucleotides can thus be employed.

A polynucleotide according to the present invention can be fused, inframe, to any other protein encoding polypeptide to encode for a fusedprotein using methods well known in the art. For example the polypeptidecan be fused to a leader sequence or a signal peptide for secretion.Similarly a T2-RNase protein can be fused (conjugated) to other proteinsusing methods well known in the art. Many methods are known in the artto conjugate or fuse (couple) molecules of different types, includingproteins. These methods can be used according to the present inventionto couple a T2-RNase to other molecules such as ligands or antibodies tothereby assist in targeting and binding of the T2-RNase to specific celltypes. Any pair of proteins can be conjugated or fused together usingany conjugation method known to one skilled in the art. The proteins canbe conjugated using a 3-(2-pyridyldithio)propionic acidNhydroxysuccinimide ester (also called N-succinimidyl 3-(2pyridyldithio)propionate) (“SDPD”) (Sigma, Cat. No. P-3415), a gluteraldehydeconjugation procedure or a carbodiimide conjugation procedure.

According to a preferred embodiment of the present invention, thepolynucleotide includes one or more segments harboring transcriptioncontrol sequences operatively linked to the T2-RNase encoding sequence.Such transcription control sequences can include, but are not limitedto, promoters and enhancers as further detailed hereinbelow. Thesetranscriptional control sequences are typically operatively linkedupstream to the coding region and function in regulating thetranscription and/or translation thereof.

According to another preferred embodiment of the present invention thepolynucleotide encoding a T2-RNase is included within a eukaryoticexpression vector. The phrase “expression vector” refers to a nucleicacid sequence which includes a sequence encoding a T2-RNase andtranscriptional control sequences and which is capable of expressing aT2-RNase within a mammalian cell.

Numerous methods for the insertion of DNA fragments into a vector, forthe purposes of mammalian gene expression are known in the art and maybe used to construct a T2-RNase encoding gene expression vectorincluding appropriate transcriptional/translational control sequencesand the desired T2-RNase polynucleotide sequences. These methods mayinclude in vitro DNA recombinant and synthetic techniques and in vivogenetic recombination. Expression of a polynucleotide encoding aT2-RNase may be regulated by transcription control sequences so that aT2-RNase is expressed in a host cell infected or transfected with therecombinant DNA molecule. For example, expression of a T2-RNase may becontrolled by any promoter/enhancer element known in the art. Thepromoter activation may be tissue specific or inducible by a metabolicproduct or administered substance.

Promoters/enhancers which may be used to control T2-RNase expressionwithin target tissues or cells include, but are not limited to, thenative RB promoter, the cytomegalovirus (CMV) promoter/enhancer(Karasuyama, H., et al., 1989, J. Exp. Med., 169:13), the human β-actinpromoter (Gunning, P., et al., 1987, Proc. Natl. Acad. Sci. USA,84:4831-4835), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (HHTV LTR) (Klessig, D.F., et al., 1984, Mol. Cell. Biol., 4:1354-1362), the long terminalrepeat sequences of Holoney murine leukemia virus (MULV LTR) (Weiss, R.,et al., 1985, RNA Tumor Viruses, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.), the SV40 early region promoter (Bemoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (RSV) (Yamamoto et al., 1980,Cell 22:787-797), the herpes simplex virus (HSV) thymidine kinasepromoter/enhancer (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), the adenovirus promoter(Yamada et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82(11):3567-71), andthe herpes simplex virus LAT promoter (Wolfe, J. H., et al., 1992,Nature Genetics, 1:379-384).

Expression vectors compatible with mammalian host cells for use ingenetic therapy of tumor or cancer cells, include, but are not limitedto, plasmids, retroviral vectors, adenovirus vectors, herpes viralvectors, and non-replicative avipox viruses, as disclosed, for example,by U.S. Pat. No. 5,174,993, which is incorporated herein by reference.

Several methods can be used to deliver the expression vector accordingto this aspect of the present invention to the target mammalian cell(s).

For example, a suitable pharmaceutically acceptable carrier such as aphysiologically appropriate solution, and which contains an effectiveamount of an expression vector can be administered topically,intraocularly, parenterally, orally, intranasally, intravenously,intramuscularly, subcutaneously or by any other effective means.

A physiologically appropriate solution containing an effective amount ofan expression vector can be administered systemically into the bloodcirculation to treat a cancer or tumor which cannot be directly reachedor anatomically isolated.

For treating tumor masses a physiologically appropriate solutioncontaining an effective amount of an expression vector can be directlyinjected, via a needle, into a target tumor mass in amounts effective totreat the tumor cells of the target tumor to mass.

Alternatively, a cancer or tumor present in a body cavity such as in theeye, gastrointestinal tract, genitourinary tract (e.g., the urinarybladder), pulmonary and bronchial system and the like can receive aphysiologically appropriate composition (e.g., a solution such as asaline or phosphate buffer, which is sterile except for the expressionvector) containing an effective amount of an expression vector viadirect injection with a needle or via a catheter or other delivery tubeplaced into the cancer or tumor afflicted hollow organ. Any effectiveimaging device such as X-ray, sonogram, or fiber optic visualizationsystem may be used to locate the target tissue and guide the needle orcatheter tube.

It will be appreciated that since a “naked” expression vector can beactively taken up by mammalian cell, uptake and targeted delivery isenhanced if the expression vector is appropriately packaged orencapsulated.

Thus, according to another preferred embodiment of the present inventionthe pharmaceutically acceptable carrier includes a delivery vehiclesuitable for the delivery of the expression vector into mammalian cellsin a targeted manner.

A viral expression vector may be introduced by a delivery vehicle into atarget cell in an expressible form by infection or transduction. Such adelivery vehicle includes, but is not limited to, a retrovirus, anadenovirus, a herpes virus and an avipox virus. A delivery vehicle ableto introduce the vector construct into a target cell and able to expressT2-RNase therein in cell proliferation-inhibiting amounts can beadministered by any effective method described hereinabove.

Alternatively, such a delivery vehicle can include, but is not limitedto, a liposome, a micelle, an antibody or a ligand as previouslydescribed hereinabove.

It will be appreciated that the polynucleotides herein described can beused in the preparation of a medicament useful in inhibiting theproliferation of a mammalian cell of a mammal, by mixing thepolynucleotide with an appropriate pharmaceutically acceptable carrier.

As already mentioned hereinabove, polynucleotides encoding a T2-RNasecan be obtained by a variety of methods, including, but not limited to,polymerase chain reaction (PCR) amplification of genomic or cDNAlibraries screening using T2-RNase specific primers, using reversetranscription PCR along with T2-RNase specific primers to amplify mRNAisolated from organisms known to express T2-RNases, or directlyisolating DNA sequences coding for a T2-RNase from the appropriateorganisms. It will be appreciated in this case that the above mentionedmethods can also be used to isolate or generate any of the active formsof a T2-RNase described hereinabove.

The purified polynucleotide can then be inserted into appropriateexpression vectors or provided with the appropriate transcriptionalcontrol sequences and prepared as described hereinabove.

As is further exemplified in the Examples section that follows andmentioned hereinabove, an assay for determining the effects of aspecific T2-RNase or a polynucleotide encoding same is also provided inaccordance with the teachings to the present invention. Such an assay iseffected, for example, by exposing proliferating cells to a T2-RNase andfollowing their proliferative behavior over time as compared to control,untreated cells. This assay can be employed not only for selecting forthe most potent T2-RNase for any specific application, but also forestablishing dose response, which can be translated into initialtreatment dosage in in vivo experiments or during treatment of asubject, all as is further exemplified herein for RNase B1 of the T2family. It will be appreciated that this assay can also be used todetermine the anti-proliferative active site or portion or a T2-RNase,or to determine the activity of generated or isolated mutants which donot display ribonucleolytic activity.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well to known in the art and are provided for the convenience ofthe reader. All the information contained therein is incorporated hereinby reference.

Example 1

Characterization of Aspergillus niger B1 Rnase and its Inhibitory Effecton Pollen Tube Growth in Fruit Trees

Materials and Methods

Preparation and Purification of A. Niger Extracellular RNase:

Aspergillus niger B1 (CMI CC 324626) was grown in liquid culturecontaining 1% (w/v) wheat flour and 0.05% (w/v) ammonium sulfate. Themixture was adjusted to pH 3.5 with hydrochloric acid and autoclaved. Aninoculum of about 10⁶ spores was suspended in 100 ml of medium andincubated at 30° C. in an orbital shaker, at 200 rpm for 100 hours. Thegrowth medium was passed through a 0.2-μm membrane and dialyzed threetimes against 10 volumes of 2 mM sodium acetate pH 6. Two liters ofdialyzed solution were loaded onto a Fractogel EMD-TMAE 650 (M) 26/10(Merck) column, equilibrated with 20 mM sodium acetate pH 6. Boundproteins were eluted with a 500-ml linear gradient of 0-1.0 M sodiumchloride in the same buffer, using a fast protein liquid chromatography(FPLC) system (Pharmacia) with a flow rate of 5 ml·min⁻¹. The fractionsexhibiting the highest RNase activity were pooled and dialyzed against 2mM sodium acetate pH 6, and a 50-ml aliquot was loaded onto a MONO-Q 5/5HR (Pharmacia) column, equilibrated with 20 mM sodium acetate pH 6. Theelution was performed as with the EMD-TMAE column, except that only 10ml of a 0-1.0 M salt gradient were used, at a flow rate of 1 ml·min⁻¹.

Proteins were monitored at 280 nm and measured according to Bradford(Bradford, M. M. 1976. Anal. Biochem. 72:248-245), using bovine serumalbumin (BSA) as a standard. Different fractions were analyzed by a12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) (Laemmeli, U. K. 1970. Nature 227: 680-685). RNase activitywas determined as previously described (Roiz and Shoseyov 1995, Int. J.Plant Sci. 156:37-41).

The purified RNase B1 was enzymatically deglycosylated according to theprocedure described by Broothaerts et al. (Broothaerts, W. P. et al.1991. Sex. Pant Reprod. 4:258-266). The enzyme was mixed with 0.5% (w/v)SDS and 5% (w/v) 13-mercapthoethanol and heated at 100° C. for 5minutes. Once cooled, the reaction mixture was diluted 2.5-fold with abuffer containing 50 mM sodium phosphate pH 7.5, 25 mM EDTA, 1% (w/v)Triton X-100 and 0.02% (w/v) sodium azide. Peptide-N-glycosidase F(PNGase F, Boehringer-Mannheim) was added to a final concentration of 20units·ml⁻¹ and incubation was performed overnight at 37° C. The samplewas then mixed with sample application buffer, heated at 100° C. for 5minutes, and analyzed by SDS-PAGE using a 12.5% gel.

RNase Assays:

The optimal conditions for RNase activity were determined according to aprocedure modified from Brown and Ho (Brown, P. H. and Ho, T. H. D. 1986Plant Physiol. 82:801-806), using a range of temperatures from 20-100°C. at 10° C. increments, and a range of pH's from 2.5-7 at 0.5 pH unitsincrements, established using 50 and 12 mM phosphate-citrate buffers.Samples of 10 μl were each added to 490 μl of ice-cold buffer,containing 4 mg·ml⁻¹ yeast RNA (Sigma). Half of each sample was used asa blank by immediately adding a stop solution containing 50 μl of 0.75%(w/v) uranyl sulfate in 25% (w/v) perchloric acid. The remaining halfwas incubated for 10 minutes, following which a 50 μl of stop solutionwas added to each. Following centrifugation at 15,000×g for 5 minutes,the supernatant was diluted 20-fold with distilled water and theabsorbance was determined at 260 nm. One unit of RNase activity wasdetermined as the amount of enzyme releasing soluble nucleotides at arate of one A.U_(260 nm) per min.

RNase B1 was visualized by an activity gel, as previously described(Roiz and Shoseyov, 1995, Int. J. Plant Scizzz. 156:37-41). An SDS gelcontaining RNase B1 was renatured by washing twice for 15 minutes eachwith 20 mM acetate buffer at pH 3.5 containing 25% (v/v) iso-propanoland then twice for 15 minutes each with buffer alone. The renatured gelcontaining the RNase B-1 was laid over a plate containing 0.1% RNA and0.8% agarose in 20 mM acetate buffer and incubated at 37° C. for 30minutes. The gel was then removed and the agarose plate was stained with0.02% (w/v) toluidine blue in water to visualize RNase activity.

The Effect of RNase B1 on Pollen Tube Growth:

Peach cv. Almog pollen was germinated in vitro in liquid culture, aspreviously described (Roiz and Shoseyov, 1995, Int. J. Plant Sci.156:37-41). Pollen grains were suspended in aliquots containing 100 μlof 15% (w/v) sucrose, 100 μg·ml⁻¹ boric acid, 200 μg ml⁻¹ magnesiumsulfate, 200 μg·ml⁻¹ calcium nitrate and different concentrations ofRNase B1. Following incubation overnight at 25° C. in a dark chamber,germination percentage was recorded. Pollen tube length was examinedwith an eyepiece micrometer.

The effect of RNase B1 treatment on pollen tube growth was also testedin vivo. Intact flowers of peach and in tangerine (Citrus reticulata,Blanco cv. Murcott) were sprayed at the early stages of anthesis with100 units·ml⁻¹ RNase B1 in 20 mM citrate buffer at pH 3.5. In eachspecies additional flowers at the same stage, on different branches,were sprayed with buffer alone or remained untreated as controls.Following exposure to open pollination for 48 hours, the styles werefixed in a 3:1 acetic acid to ethanol (by volume) for 24 hours, washedwith distilled water and imbibed overnight in 8 M sodium hydroxide.Following thorough washing in distilled water, the styles were cutlongitudinally, immersed each in a drop of 0.1% (w/v) aniline blue in0.1 M potassium phosphate on a slide and carefully squashed with a coverglass. Pollen tubes were observed by epifluorescence microscopy (OlympusBX40 equipped with WIB cube).

The Effect of RNase B1 on Fruit Set:

Field experiments were done in nectarine (Prunus persica var. NectarinaFantasia). Branches of 30-40 cm long, bearing approximately 10% openflowers, were sprayed with different concentrations of RNase B1 in 20 mMcitrate buffer pH 3.5 and 0.025% triton-X 100. Untreated branches, andbranches sprayed with only buffer and triton-X 100, served as controls.The branches were sprayed at 2- to 3-days intervals during the bloomingperiod (14 days). A month later, the number of fruit per branch wasexamined. For viability test, seeds were cut longitudinally through theembryo and immersed in 1% 2,3,5-Triphenyl tetrazoluim chloride in waterfor 4 to hours at 20° C. in a dark room. Red staining indicated viabletissues.

Experimental Results

Purification and Characterization of RNase B1:

A. niger grown in liquid culture produced considerable amounts ofextracellular RNase B1. A temperature of 60° C. and a pH of 3.5 werefound optimal for RNase activity, and were adopted as the standardconditions for subsequent RNase assays.

RNase B1 purification included three steps (Table 4). In a first step acrude filtrate contained 1000 units·ml⁻¹ and 0.05 mg·ml⁻¹ protein wasobtained. The crude filtrate was passed through an EMD-TMAE column andthe pooled active fractions (FIG. 1, graph A) contained 0.1 mg·ml⁻¹protein, with an RNase activity of 40,000 units·ml⁻¹. In the final step,the pooled fractions were passed through a MONO-Q column and the activeRNase fraction was eluted (FIG. 1, graph B). This fraction contained aprotein concentration of 1.05 μg·ml⁻¹ and RNase activity of 543,000units·ml⁻¹. Two major protein bands, of 40 and 32 kDa, were observedfollowing SDS-PAGE of the purified RNase B1 fraction (FIG. 2). An RNaseactivity gel showed active bands corresponding to the 32 and the 40 kDaproteins. When subjected to PNGase F, a single protein band appeared at29 kDa. RNase activity was retained after PNGase digestion (not shown).

TABLE 4 Specific Protein activity Total concentration Recovery (units/mgPurification step units (mg/ml) (%) protein) Crude filtrate 2,000,0000.05 100 20,000 EMD-TMAE 1,120,000 0.1 56 400,000 column MONO-Q 652,2001.05 32.6 517,143 column

The Effect of RNase B1 on Pollen Tubes and Fruit Set:

In in vitro, experiments 75% of the control pollen grains germinated andthe pollen tubes reached about 0.5 mm in length. Addition of RNase B1 tothe growth medium reduced the percentage of germination and the lengthof the pollen tubes, in a dose responsive manner (FIG. 3). RNase B1 hada pronounced inhibitory effect, 50 units·ml⁻¹, representing 0.1 μg·ml⁻¹protein, were lethal, whereas 125 μg·ml⁻¹ of BSA reduced only half ofpollen germinability and tube growth.

In vivo, control pollen tubes of peach were observed growing through thestigmatic tissue directed into the style 48 hours after pollination(FIG. 4 a). A similar effect was observed in styles treated with bufferonly. In contrast, pollen grains germinated on stigmas treated withRNase B1 produced short pollen tubes, which appeared to lack any growthorientation, and failed to penetrate the stylar tissue (FIG. 4 b). Intangerine only a small portion of the stigmatic tissue, the diameter ofwhich was 2-3 mm, was captured by the view field of the microscope.Therefore, only few pollen tubes were observed, as shown in FIG. 5.However, the difference between the normal growth of the control pollentubes (FIG. 5 a) and the irregular growth of the RNase-treated pollentubes (FIG. 5 b), was clearly evident.

In nectarine cv. Fantasia, RNase B1 caused a reduction in fruit set(Table 5). In branches that remained untreated or sprayed with bufferwith triton X-100, fruit set was 48.3% and 36.3%, respectively. Itseemed that the low pH-buffer had some inhibitory effect on fruit set,however branches treated with 500 and 1000 units·ml⁻¹ of RNase B1 set23.3% and 18.4% fruits, respectively, indicating a significant thinningeffect of the RNase, in a dose dependent manner.

TABLE 5 Treatment Flowers (total number) Fruit set (%) Control untreated169 48.3 a* Control buffer 143 36.3 ab 500 units/ml RNase B1 148 23.3 bc1000 units/ml RNase B1 106 18.4 c *values not sharing a common letterare significantly different at P = 0.05.

In RNase B1 treated branches many undeveloped fruitlets were observed.Viability tests showed that in the control flowers (either untreated orsprayed with buffer only), embryo tissues were stained red, (FIG. 6 a),whereas the tissues of embryos developed in RNase-treated flowers,stained brown indicative of necrosis (FIG. 6 b).

Aspergillus niger B1 extracellular RNase (RNase B1) was purified to tohomogeneity. It was found to contain two isoforms of 32- and 40-kDaglycoproteins, sharing a 29-kDa protein core. The optimal RNase activitywas observed at a temperature of 60° C. and a pH of 3.5. In peach(Prunus persica cv. Almog) and tangerine (Citrus reticulata, Blanco cv.Murcott) the enzyme inhibited pollen germination and tube growth invitro as well as in vivo. In field experiments, the RNase caused areduction in nectarine (Prunus persica var. nectarina Fantasia) fruitset and inhibited normal embryo development.

Example 2 Inhibition of Pollen Germination and Tube Growth by T2-RNaseis Mediated Through Interaction with Actin

The inhibition of pollen germination and tube growth by RNase is wellrecognized, yet the mechanism by which this enzyme interferes with theelongation process is still unclear. As such, this study set out todecipher the role of RNase B1 in interfering with the elongation processof pollen tubes.

Materials and Experimental Methods

The Effect of RNase B1 on Pollen Tubes Growth:

Anthers of lily (Lilium grandiflorum L. cv. Osnat) were let to dehiscefor 24 hours at room temperature and than either used fresh or stored at−20° C. RNase B1 was produced and purified from Aspergillus niger growthmedium filtrate as described in Example 1. Pollen was germinated invitro in aqueous cultures of 100 μl each, containing 7% sucrose, 1.27 mMCaNO₃, 0.16 mM H₃BO₃, 1 mM K₂NO₃ and 3 mM KH₂PO₄ in water (Yokota andShimmen 1994). Some cultures were supplemented with RNase B1 having 100units/ml of RNase activity to a final protein concentration of 16 μg/ml.Additional cultures were supplemented with RNase that was previouslyboiled for 30 minutes which produced the loss of 50% of activity, orwith autoclaved RNase lacking any catalytic activity. Following 2 hoursof incubation at 25° C. in the dark, pollen tube length was measuredunder the microscope eyepiece micrometer. The pollen tubes were stainedwith IKI (0.3% I₂ and 1.5% KI in water) to detect starch bodies.

Actively extending 1-hour pollen tubes were transferred to glass cellson the microscope stage. The pollen tubes growth pattern and organellemovement were video recorded as modified from Heslop-Harrison andHeslop-Harrison (Heslop-Harrison, J. and Heslop-Harrison, Y. 1990. SexPlant Reprod. 3:187-194), using Applitec MSV-800 video presenter. Imageswere captured at 0.8 frames/sec for 8 seconds by Scion LG-3 framegrabber and then digitized and integrated by NIH image software. Thephotographs were processed using Adobe Photoshop (Adobe Systems Inc.,Mountain View, Calif.) and Power-Point (Microsoft Co.) softwares.

The Effect of RNase on Pollen Tube Actin Filaments:

Pollen was germinated in vitro in aqueous cultures with or withoutRNase.

Following incubation overnight, the pollen tubes were gently pelletedand the growth medium was replaced with 10⁻⁶ M tetramethylrhodamine Bisothiocyanate (TRITC)-labeled phalloidin (Sigma) in PBST buffer (150 mMNaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ and 0.02% Tween-20). For invivo observations, lily cv. Stargazer flowers were emasculated at theonset of anthesis and 0.5 ml of a growth medium containing 100 units/mlRNase were injected through the stigma into the stylar canal. Flowersinto which growth medium without RNase was injected were used as acontrol. The liquids were absorbed into the stylar tissue for 5 hours at25° C., following which the stigmas were hand-pollinated by lily cv.Osnat pollen. Following 48 hours of incubation at 25÷C, each pistil wascut longitudinally and the pollen tubes were carefully excised andremoved into TRITC-TBST solution and incubated for 1 hour. The incisionat the stigma did not affect the pollen tubes, since their vitalprotoplasts were located at the distal part, protected by callose plugs.In both the in vitro and in vivo experiments the stained pollen tubeswere rinsed in TBS (TBST lacking Tween-20), placed on a glass slide andobserved with an epifluorescent light microscope (Olympus BX40 equippedwith USH-102D mercury to lamp).

Binding of Actin to RNase B1:

The interaction between RNase B1 and actin was quantified as modifiedfrom Simm (Simm, F. C. et al., 1987. Eur. J. Biochem. 166:49-54). Rabbitmuscle globu(G-) actin (Sigma Co.) was polymerized to filamentous (F-)actin in Buffer F (10 mM Tris pH 8, 0.1 mM ATP, 0.2 mM CaCl₂, 0.1 M KCLand 2 mM MgCl₂) for 30 minutes at room temperature. Samples of 50 μlcontaining each 30 μM F-actin were incubated overnight at 4° C. with1-33 μM RNase B1. As a control, each concentration of RNase wasincubated with buffer F alone. The samples were centrifuged at 15,000 gfor 40 minutes and RNase activity of the supernatant was determined(Roiz, L., Goren, R. and Shoseyov, 0.1995, Physiol. Plant. 94:585-590.).

Immunogold Silver Staining of RNase B1 on Pollen Tubes:

Immunogold silver stain (IGSS) was used to detect RNase attachment tolily pollen tubes. Polyclonal antibodies were raised against RNase B1 inrabbit (Aminolab). In vitro 2-hours lily pollen tubes were fixedovernight in 2.5% gluteraldehyde in PBST at 4° C. The pollen tubes werewashed for 1 hour in PBST, blocked for 1 hour in PBST containing 1% BSAand 2% skim-milk and incubated for 1 hour in anti-RNase B1, diluted1:500 in PBST. Rabbit pre-immune serum (PIS) was used as control. Thepollen tubes were washed three times, 10 minutes each, in PBST and thenincubated for 1 hour in goat anti-rabbit IgG conjugated with 5-nm goldparticles, diluted 1:100 in PBST. Following two 10 minutes-washes inPBST and one 10 minutes-wash in water, a silver-stain kit (BioCellResearch Laboratories) was used for the final development of thereaction. The pollen tubes were soaked in the combined kit solutions for10-15 minutes, washed in excess distilled water and observed under alight microscope (Olympus BX40).

Experimental Results

A control sample of lily pollen tubes, germinated in vitro in a growthmedium without RNase, reached about 300 μm in length (FIG. 7). Culturesthat were treated with RNase under the same conditions, reached only 160μm in length. Boiling or autoclaving the RNase yielded pollen tubes of130 and 170 μm in length, respectively. The differences between thethree RNase-treated groups of pollen tubes were deemed to insignificant.

Starch-staining showed that amylioplasts of the control sample wereobserved spreading along the pollen tube, except at the tip zone (FIG. 8a). On the other hand, in RNase-treated pollen tubes IKI-stained bodiesaccumulated at the tip zone (FIG. 8 b).

The integrated video images of actively extending pollen tubes displayedthe cytoplasmic flow lines (FIGS. 9 a and 9 b). In the control sample acontinuous longitudinal movement characteristic of normal pollen tubedevelopment was most common, as was acropetal flow at the tube peripheryand basipetal flow at the center, forming an “inverse fountain” patternbeneath the tip zone (FIG. 9 a). The tip zone itself was occupied bymuch smaller bodies, mainly P-particles, the movement pattern of whichwas hardly observed. In RNase-inhibited pollen tubes the disruption ofactin filament assembly is evident. The stunted growth tip appearedswollen, with well-visible starch and lipid particles reaching the tipzone (FIG. 9 b). No continuous movement could be detected, but insteadextended irregular images indicated cytoplasmic bodies rotatingrandomly.

The effect of RNase on the actin filaments distribution was examined in1-hour in vitro and 48-hours in vivo pollen tubes. The in vivo pollentubes reached about 3-4 cm long, and their TRITC-phalloidin staining wasmore intensive than in the in vitro tubes. However, the mode of theRNase effect was similar in both experiments. In the control, actinmicrofilaments were assembled longitudinally along the tube axis,forming a fine network in the tip zone (FIG. 10 a). On the other hand,in RNase-treated pollen tubes masses of actin were accumulated at thetip cell wall (FIG. 10 b).

The interaction between RNase B1 and actin was quantified usingScatchard analysis. In the actin-RNase B1 binding experiment aregression line, intersecting with the abscissa at 0.45 (FIG. 11)indicated that the RNase:actin molar ratio was 0.45, implying that twoactin molecules bind to each RNase molecule.

Pollen germinated in the presence of RNase B1 were prepared for lightmicroscopy and the location of RNase was determined by IGSS, usinganti-RNase antibodies (FIGS. 12 a-c). In pollen tubes grown withoutRNase (FIG. 12 a) or with to RNase but treated with PIS (FIG. 12 b), thecell wall external surface was devoid of silver staining. On the otherhand, in pollen tube treated with RNase B1, a clear immunogold silverstain appeared, accumulating upon the tip zone (FIG. 12 c).

In this study Lily (Lilium grandiflorum) pollen germination and tubeelongation were specifically inhibited by A. niger RNase B1. Boiled orautoclaved RNase, lacking most of the original catalytic activity,showed a similar inhibitory effect. The results demonstrate that A.niger RNase is a protein having an actin-binding activity clearlyrelated to the inhibitory effect of the T2 RNase on pollen tubeelongation. This actin binding activity, which is unrelated to thecatalytic activity of RNase B1, deforms the pollen tube actin filamentsarrangement to thereby disrupt cytoplasmic streaming, cell motility andgrowth.

Example 3 The Effect of RNase B1 on Human Colon Cancer Cells

Since the actin binding activity uncovered for RNase B-1 in pollen tubeshinted at a possible cytotoxic activity it was decided to examine thecytotoxic effect of RNase B1 on human colon cancer cells.

Materials and Experimental Methods and Results

Cell Culture:

All the experiments were performed in vitro. Human colon adenocarcinoma(HT29) cells were grown in DMEM medium (Biological Industries, BetHaemek), supplemented with 10% fetal calf serum, 1% glutamine and 10%Antibiotic-Antimicotic solution (Biolab). The cells were incubated at37° C. in a humidified atmosphere containing 5% CO₂. RNase B1 solutionswere made in PBS buffer, pH 6.8.

Preliminary Cell Viability Assay:

Cells were incubated with 50-ml flasks. Each flask contained 2×10⁵ cellsin 7 ml medium, in the absence or presence of different concentrations(10⁻⁸-10⁻⁶ M) of RNase B1. The cells were grown for 48 hours or 72hours, and then viable and non-viable cells were differentially countedusing trypan blue staining.

In all treatments the total number of cells grown for 72 hours(55-60×10⁵) exceeded to by about twofold the number of cells obtainedafter 48 hours culture (25-30×10⁵) (FIG. 13 a). The presence of RNase B1in the growth medium did not have a significant effect on cell growth.However, a small but significance effect of the RNase B1 on the numberof dead cells was found at both 48 hours and 72 hours of incubation(FIG. 13 b).

Clonogenicity Assay I:

The long-term survival of tumor cells is characterized by their abilityto divide and produce clones. Cells were preincubated for 48 hours ingrowth medium containing 10⁻⁶ M RNase B1, then trypsinized, washed andresuspended in growth medium lacking RNase B1. Prior to plating into96-well microtiter plates, the cells were diluted to serial 5-folddilutions ranging between 50-to-10⁵ cells in each well (200 ml). Theplates were incubated for 14 days in the conditions described above,without adding fresh growth medium following which the colonies werefixed and stained with methylene blue. The clonogenic cells in each wellwere numbered following clones visualization. Control cells were treatedas above, but preincubated during the first 48 hours in medium lackingRNase B1.

In both treatments a similar number of colonies was observed in wells inwhich 100 cells were plated (FIG. 14). The cytotoxic effect of the RNaseB1 appeared in wells containing higher densities of cells. In wellsplated with 500 cells each, the control and the RNase B1-treated cellsproduced 180 and 100 colonies per well, respectively. Furthermore, inwells plated with 1000 cells each the RNase B1-treated cells formedabout 250 colonies per well, whereas the control cells formed numerouscolonies that fused to a continuous layer, thus could not be counted andillustrated in FIG. 14. Cells plated at higher densities did not survivethe culture without changing media.

Clonogenicity Assay II:

The ability of the tumor cells to proliferate and to colonize wasexamined in short vs. continuous exposure to RNase B1. The experimentwas performed as described in Clonogenicity assay I using (i) controlcells, (ii) cells preincubated with medium containing 10⁻⁶ M RNase B1and then allowed to colonize in RNase B1-free growth medium, and (iii)cells preincubated as in (ii) and then incubated during the colonizationassay in growth medium containing 10⁻⁶ M RNase B1. In these experimentsthe initial densities ranged between 250-1000 cells per well and thecolonization period was 7 days.

The shorter period of incubation used in this experiment (7 days)compared to a 14 day preincubation resulted in non defused colonies,which could be distinguished even in wells containing high densities ofcells. In all densities, 48 hours of preincubation in RNase B1 led to areduction of 20-30% in the ability of the cells to colonize, compared tothe control (FIG. 15). However in each density a continuous exposure toRNase B1 led to a dramatic reduction of 90% in clonogenicity. FIGS. 16a-c Show that the continuous RNase B1-treated cells (FIG. 16 c) weresmaller and less stainable than the cells that were preincubated for 48hours in RNase B1 (FIG. 16 b) or the control cells (FIG. 16 a). Thisresult indicate that RNase B1 affected the colonies growth rate.

Thus as is clearly shown from the results presented herein A. nigerRNase B1 has a clear cytotoxic effect on human adenocarcinoma HT29cancer cells. The cytotoxic effect of RNase B1 is expressed viareduction of cell clonogenicity, rather than reduction of cellviability. It is possible that the RNase B1 has a long-term effect onthe tumor cells. The RNase B1 causes a reduction in the colonies growthrate compared to the control, indicating that it may affect the abilityof the cells to proliferate.

Example 4 The In Vivo Effect of RNase B1 on Tumor Development in a RatModel

For further studying the anti-cancer effect of RNase B1, an in vivoexperiment was conducted in rats.

Materials and Experimental Methods

Charles-River derived 4-weeks old male rats were divided into groups of6. In some groups, the rats were induced to develop colon cancer by fiveweekly injections of Dimethylhydrazine (DMH). In this experiment, twomodes of RNase B1 administration were examined. RNase B1 was applieddirectly into the colon by osmotic micro-pumps, or given orally usingenterocoated microcapsules. The full set of treatments each rats groupreceived is described by the scheme of FIG. 1. During the experiment,the rats were weighted weekly to monitor the effect of DMH and/or RNaseB1 on their growth rate. In groups treated with RNase B1 feces wascollected weekly from each cage and dried at 60° C. A sample of 250 mgdry feces was grounded and re-dissolved in phosphate buffer saline(PBS). Following centrifugation, RNase activity in the upper solutionwas examined as described in Roiz et al. (Roiz, L. et al., J. Amer. Soc.Hort. Sci. 125(1):9-14. 2000).

Administration of RNase B1 to the Colon Via Osmotic Micro-Pumps:

RNase B1 was loaded into osmotic micro-pumps (ALZET). The pumps wereimplanted subcutaneously into the rats abdomen. The osmotic pumpsallowed a constant release of RNase B1 directly into the colon via acatheter, at a calculated concentration of 10⁻⁶ M in the colon for atleast 6 weeks, assuming that the rat colon is about 4 ml in volume. Ratswere treated as follows: Rats of one group were implanted with pumpscontaining “live” RNase B1 (RNase B1), having full RNase activity. Ratsof a second group were implanted with pumps containingautoclave-inactivated RNase B1 (1-RNase B1), lacking any RNase activity.Whereas, rats of a third group were implanted with pumps containing PBS,which was used as the RNase B1 vehicle in the first two groups, andserved as controls.

To examine a possible preventive effect of RNase B1, selectedDMH-treated rats received RNase B1-9 weeks after the first DMHinjection. Then, the rats were sacrificed and their colons excised andwashed with PBS and then with PBS containing 0.1 M dithiothreitol (DTT).The colons were thereafter opened longitudinally and fixed for at least1 hour in 4% formaldehyde in PBS over a filter paper. Following stainingwith 0.025% methylene blue in PBS, the colon mucosa was observed via amicroscope under low magnification for aberrant crypt foci (ACF). ACFwere counted in the distal (5 cm) colon.

To examine a therapeutic effect of RNase B1, the rest of the ratsreceived RNase B1 from 12 to 17 weeks after first DMH injection. Thecolons were excised and fixed as described above and the tumors countedand measured. For histopathological examinations, each tumor wasembedded in paraffin. Thin (10 μm) sections were stained and the degreeof malignancy was evaluated.

Oral Administration of RNase B1:

Preparation of Microcapsules:

Microcapsules were prepared with a modified procedure described by Linet al. (Lin J. J., et al., 1994, Biochem. Biophys. Res. Commun. 14;204(1):156-62). A mixture of 0.6 grams lyophilized RNase B1 and 2.4grams glucose was well powdered by a mortar and pestle. The fine powderwas poured into a 1 L beaker containing 200 ml liquid paraffin and 2 mlSpan-80 and stirred at 600 rpm for 20 min. Acetone-ethanol-celluloseacetate phthalate (CAP) solution was carefully added to the abovestirring mixture (3.2 grams CAP in 40 ml of 9:1 acetone: 95% ethanol)and let stir for further 2 h in a hood, to remove traces of acetone. Themicrocapsules were hardened by adding 30 ml ether and dried on filterpaper using Buchner funnel and traces of liquid paraffin were removed bytwo additional washes with 30 ml ether. The microcapsules were then letdry overnight and pass through a fine mesh. Most microcapsules werebetween 200 and 500 μm.

In a preliminary experiment (FIG. 18), CAP microcapsules were foundinsoluble in acidic pH, representing the stomach environment. However,after 1 hour in alkaline pH maximum RNase activity was reached,indicating that the microcapsules could easily release their content inthe intestines.

Oral Administration of the RNase B1 Microcapsules:

The microcapsules containing RNase B1 or glucose as placebo, were mixeswith ground Purina chows. Each RNase B1-treated rat received a dailydose of 1.6 mg RNase B1, to gain a final concentration of 10⁻⁵ M in thecolon. The experimental details for oral administration were asdescribed above for the micro-pumps administration, except for a delayedtermination (11 weeks) while evaluating the preventive effect of RNaseB1 (FIG. 17).

Experimental Results

The Effect of the Different Treatments on Rat Growth Rate:

The rats initial weight was about 200 grams. The experiment ended atdifferent time for each treatment, as described above. Generally, therats reached a final body weight of about 400-500 grams, with nosignificant differences between the different treatments (FIG. 19 a-d).However, DMH-treated groups showed a slight decrease in to body weightcomparing to RNase B1-treated groups, in the presence or absence of DMH.

RNase Activity in Rat Feces:

FIGS. 20 a-c show changes in RNase activity in feces of rats implantedwith osmotic pump containing RNase B1, I-RNase B1 or PBS in a preventivemode (FIG. 1) during 8 weeks. In rats treated with RNase B1 (FIG. 20 a)RNase activity in feces was 5-fold higher than in rats treated withI-RNase B1 (FIG. 20 b) or with PBS (FIG. 20 c). This activity wasmaintained high for 5 weeks and then decreased gradually, as thereservoir of RNase B1 in the pumps exhausted. A basal endogenous RNaseactivity was detected in feces of the two latter groups.

Similar pattern was observed in rats fed with microencapsulated RNaseB1, but an 8-fold higher RNase activity was detected as is compared torats fed with microcapsules containing glucose only (FIG. 21). In thisexperiment, the gradually decrease in RNase activity may be explained asa result of increase in the rats body weight and as a consequence, thecolonic volume.

The Effect of RNase B1 as a Preventive Agent:

The pump-implanted rats were sacrificed after 8 weeks, since infectionat the pump sites was observed. At this stage only ACF were apparent.ACF are surrogated biomarkers of carcinogenic changes in the rat colonduring the initiation phase of carcinogenesis. Goblet cells in cryptaebecome larger and were intensively stained comparing to the normalmucosal cells. ACF counts were dramatically reduced due to RNase B1 orI-RNase B1 treatments (FIG. 22). No damaging effect on colon mucosa wasobserved in rats treated with RNase B1 or I-RNase B1 in the absence ofDMH.

In rats fed with microencapsulated RNase B1, the experiment continuedfor 11 weeks following the first DMH administration. At that time, bothtumors and ACF were present. RNase B1 caused a reduction in the numberof tumors per colon FIG. 23 a), in tumor size (FIG. 23 b) as well as inthe number of ACFs (FIG. 23 c) as is compared to control.

In addition, a color diversity of colon tumors was observed; red tumorsthat had intensive blood supply (FIG. 24 a), white tumors almost devoidof blood vessels (FIG. 24 b), and “pink” tumors with only few bloodvessels (FIG. 24 c). In glucose-treated rats all the tumors were red. Onthe other hand, in RNase B1-treated rats a significant reduction in thenumber of reddish tumors was observed (FIG. 24 d); 10% and 50% of thetumors were pink and white, respectively. These results clearly indicatean anti-angiogenic effect of RNase B1.

Tumors could also be distinguished by histopathological parameters, asbenign or malignant. A benign tumor termed adenoma (FIG. 25 a) can bedefined by a propagated mucosal layer, and sometimes by development ofadenopapilloma, however the submucosa is intact and well distinguishedfrom the other colon layers. In a malignant tumor, termed adenocacinoma,mucosal cells penetrate beneath the submucosa and eventually lead to aloss of tissue arrangement (FIGS. 25 b and 25 c). Examinations of thedistribution of the different types of tumors in rats treatedpreventively with glucose or RNase B1 showed that RNase B1 clearlyreduced the degree of malignancy (FIG. 9 d).

The Effect of RNase B1 as a Therapeutic Agent:

In both modes of application, either directly by osmotic pumps ororally, the well-developed tumors were exposed to RNase B1 during weeks12-17 of the experiment. In rats treated by osmotic pumps, RNase B1caused a reduction in the number of tumors per colon (FIG. 26 a). Theinhibitory effect, about 50% relative to the control, was mostsignificant in rats treated with I-RNase B1.

RNase B1 affected also tumor growth, as demonstrated by sizedistribution (FIG. 26 b). In general, most tumors had a diameter of 3-5mm, however in PBS-treated rats exceptionally large tumors, of more than9-12 mm, appeared. This result implies that RNase B1 inhibits or arreststhe development of pre-existing tumors. Nonetheless, no significantdifferences between the effects of RNase B1 and I-RNase B1 wereobserved.

As in the experiment of RNase B1 preventive effect, the pattern ofangiogenesis was also affected by RNase B1 applied via osmotic pumps(FIG. 26 c). In PBS-treated rats most of the tumors, about 80%, were redin color. In contrast, in both RNase B1 and I-RNase B1 treated rats only30% of the tumors were red, whereas the other were pink or white. Itappears that RNase B1 reduces angiogenesis also in pre-existing tumors.

In rats fed with encapsulated RNase B1, the effect of the treatment wasless significant than that obtained by osmotic pumps (FIGS. 27 a-c).This result is explained by assuming that a very small proportion of theprotein reaches to the colon. As mentioned before, the microcapsulesindeed pass the stomach, but they still have a long route through thesmall intestine and the cecum. An experiment to test this hypothesis wastherefore conducted using CAP microcapsules loaded with a fluorescentprotein and given to rats. The rats were sacrificed after 6 hours andthe content of their gastrointestinal tract was observed under afluorescent microscope. It was found that in the duodenum themicrocapsules started to dissolve. The dissolution further processed inthe ileum and jejunum. When the microcapsules reached into the cecum,most of the fluorescence was diffused into the cecum content. Since themicrocapsules were damaged, RNase B1 effect may be reduced due toproteases present in the intestine and cecum.

Despite the fact that orally administered RNase B1 did not decrease thenumber and size of pre-existing tumors, the distribution among tumortypes of color was slightly affected (FIG. 27 c). Glucose- and RNase B1treated rats had about 60% and 40% red tumors, respectively. It appearsthat orally administrated RNase B1 in the present formulation alsoaffect angiogenesis, but in a moderate way.

Example 5 The Effect of RNase B1 on Human Ht-29 Colon Cancer Cells InVitro Material and Experimental Methods

Cell Growth Conditions:

All experiments were performed in human colon adenocarcinoma (HT-29)cells. The cells were grown in 50-ml flasks containing DMEM medium(Biological Industries, Bet Haemek), supplemented with 10% fetal calfserum, 1% glutamine and 1% Antibiotic-Antimicotic solution (Biolab). Thecells were trypsinizated, and 2 ml medium containing 5×10⁴ cells wereplated in each well of a 6-wells plate. Some to plates were supplementedwith RNase B1, to a final concentration of 10⁻⁶ M. The cells wereincubated at 37° C. in a humidified atmosphere containing 5% CO₂. After48 hours the medium in the presence or absence of RNase B1 was replacedin each well respectively, to maintain a constant supply of ingredientsand RNase B1. After four days the medium was removed and the cellcultures were fixed in 4% formaldehyde in PBS (150 mM NaCl, 3 mM KCl, 10mM Na₂HPO₄, 2 mM NaH₂PO₄) pH 7.2 for 15 mM on ice. The cells werestained aiming different purpose, as follows.

Direct Staining for Intracellular Actin:

The cells were washed in PBS and permeabilized in PBS containing 0.02%Tween-20 (PBST) for 1 hour in room temperature. Following 3 washes withPBS, the cells were stained for actin with 10⁻⁶ M tetramethylrhodamine βisothiocyanate (TRITC)-labeled phalloidin (Sigma) for 1 hour and letstand in PBS overnight at 4° C. to remove any excess of stainingmaterial. The cells were spread in water on a glass slide and visualizedusing Confocal Laser Scanning Microscope (LSM) 510 (Zeiss).

Immunostaining for Membranal Actin:

The cells were fixed with formaldehyde and washed with PBS as describedabove, and then incubated with rabbit anti-actin antibodies (Sigma)diluted 1:500 in PBS for 1 hour at room temperature and washed threetimes with PBS. The cells were then incubated with goat anti-rabbit IgGconjugated to fluorescein isothiocyanate (FITC) diluted 1:100 in PBS foranother 1 hour at the same conditions, washed again and visualized asdescribed above.

Immunostaining of RNase B1 on Cell Surface:

Polyclonal antibodies (Aminolab, Israel) were raised in rabbit againstpurified RNase B1. Anti-RNase B1 was used as primary antibody in HT-29immunostaining, according to the procedure describe above.

Experimental Results

Direct Staining for Intracellular Actin:

In control cells growing without RNase B1, a fine actin network wasobserved stained with TRITC, filling the cell cytoplasm. A light stainwas observed at the membrane surface (FIG. 28 a). In contrast, in RNaseB1-treated cells the membrane and the peripheral zone of the cytoplasmin each cell was intensively stained (FIG. 28 b), indicatingrearrangement of actin network as a response to external addition ofRNase B1.

Immunostaining for Membranal Actin:

The FITC-immunostaining showed fine fluorescent spots of actin at themembranal zone in control cells (FIG. 29 a). This result coincides withthe TRITC staining shown in FIG. 28 a. In this experiment, no detergentwas used, thus the antibody hardly penetrated into the cells cytoplasm.Therefore, it appears that in these cells, membranal actin interactswith the external environment. In RNase B1-treated cells a much weakerimmunostaining was observed (FIG. 29 b), implying that RNase B1previously bound to membranal actin interfered with the binding of theanti-actin antibodies.

Additional cells, not treated with RNase B1, were incubated with thepreviously mixed rabbit anti-actin and 1 μM actin. A similar faintfluorescence was observed, as described in FIG. 29 b (not shown). Toeliminate the possibility of spontaneous FITC fluorescence, cells weretreated as describe, except anti-actin was omitted. No fluorescentstaining was detected.

Immunostaining of RNase B1 on Cell Surface:

Very faint FITC-fluorescence appeared in control cells incubated withanti-RNase B1 (FIG. 30 a). However, RNase B1-treated cells exhibited anintense fluorescent response (FIG. 30 b). This result indicates asignificant presence of RNase B1 over the cell surface, especially onthe cells edges and extensions. Treatment with rabbit pre-immune serum(PIS) instead of anti RNase B1 resulted in a very faint fluorescence(FIG. 30 c).

Example 6 The Effect of IAc-RNase B1 on Lily Pollen Tubes GrowthMaterials and Experimental Methods

Iodoacetylation of RNase B1:

Iodoacetylation of RNase B1 was performed according to Irie et al (IrieM, et al. 1986 J. Biochem. 99(3):627-33). RNase B1 was dissolved in 2.5ml of 0.1 M acetate buffer, containing 0.1 M iodoacetate, to a finalconcentration of 10 nM. Following incubation overnight at 37° C., theprotein was desalted on a Sephadex G-15 column. The fractions containingthe protein were collected and intensively dialyzed against water. Afterlyophilization, 10 mg of protein were obtained. The iodoacetylated(IAc-) RNase B1 RNase activity was compared to the non-modified RNaseB1.

The Effect of IAc-RNase B1 on Lily Pollen Tubes:

Lyophilized IAc-RNase B1 was dissolved to a final concentration of 1 or5 μM in lily pollen tube growth medium containing 7% sucrose, 1.27 mMCa(NO₃)₂, 0.16 mM H₃B03, 1 mM KNO₃ and 3 mM KH₂PO₄ in water (Yokota, E.and Shimmen, T. 1994. Protoplasma 177: 153-162). Lily (Liliumlongiflorum) pollen grains were germinated in vitro in tubes containing100 μl of growth medium, in the presence or absence of 10⁻⁶ M IAc-RNaseB1. As additional controls, lily pollen was germinated in the sameconcentrations of RNase B1 or BSA in growth medium. Following 1.5 hourincubation at 25° C. in darkness, pollen tube length was measured ineach treatment under light microscope.

Experimental Results

Iodoacetylation of RNase B1:

Iodoacetate leads to the inhibition of RNase activity, via binding tohistidine residues in the active site of RNase. RNase activity ofIAc-RNase B1 was 90% less, compared with non-treated RNase B1.

The Effect of IAc-RNase B1 on Lily Pollen Tubes Growth:

Control lily pollen tubes reach a length of 0.26 mm (FIG. 31). In thisexperiment, BSA was used as a control, since it does not have anycytotoxic effect on pollen tubes. Indeed, at a concentration of 10⁻⁶ M,BSA did not have a significant effect on pollen tube growth. Theinhibitory effect of 5×10⁻⁶ M BSA may be explained as a result of thefact that pollen tubes are sensitive to changes in the growth mediumosmotic potential. Both RNase B1 and IAc-RNase B1 exhibited a clearinhibitory effect on pollen tube growth. In both concentrationsIAc-RNase B1 was more effective than RNase B1, however the differenceswere not significant. Thus, in lily pollen tubes, IAc-RNase B1 inhibitspollen tubes growth in a similar manner of non-modified RNase B1,showing that loss of RNase activity does not reduce its inhibitoryeffect.

Example 7 Members of the T2 RNase Family Exhibit Anti-Angiogenic andAnti-Cancer Properties

Ribonucleases of the T2 family have been shown to share fundamentalcharacteristics, e.g. complete homology of catalytic active site,optimal RNase activity at high temperature and low pH, molecular weightof at least 24 kDa (twice that of RNase A family) and the presence ofglycan chains. To determine if members of the T2 RNase proteins haveanti-angiogenic properties the human umbilical vein endothelial cell(HUVEC) tube formation assay was employed, as follows.

Materials and Methods

The human umbilical vein endothelial cell (HUVEC) tube formationassay—Generally, the experiment was conducted as previously describedfor RNase B1 in Ponoe, M. L., 2001 (In vitro matrigel angionesis assays.In Murray, J. C. (ed.) Methods in molecular medicine: Angiogenesisprotocols. Humana Press, Totowa, N.J., vol. 46, pp. 205-209). Briefly,Freshly isolated human umbilical vein endothelial cells (HUVEC) weremaintained in M199 medium supplemented with 20% FCS, 1% glutamine, 1%antibiotic-antimycotic solution, 0.02% ECGF and 50 U/100 ml heparin.Wells of 96-well plates were coated with growth factor-depletedMatrigel| (Ponoe M L. 2001. In: Murray J C, editor. Methods in molecularmedicine. Vol. 46: Angiogenesis protocols. Totowa (NJ): Humana Press. p.205-9) in M199 media containing 5% FCS and 0.005% ECGF, supplementedwith 1 μg/ml angiogenin (to induce tube formation), and were plated with14,000 HUVECs per plated/well. The various RNases i.e., Aspergillusniger RNase B1, Aspergillus oryzae RNase T2 (Sigma, 29 kDa) or E. coliRNase I (Ambion, 27 kDa) were added at a final concentration of 2 μM.For controls, cells were plated under the same conditions, in theabsence or in the presence of any of the above RNases, but in theabsence of angiogenin. The 96-well plate was incubated for 24 hours at37° C. under a humidified atmosphere and 5% carbon dioxide. Threereplications were performed for each treatment.

Experimental Results

RNase B1 exhibits a significant anti-angiogenic effect on the HUVEC tubeformation—The capacity of various RNases (i.e., Aspergillus niger RNaseB1, Aspergillus oryzae RNase T2 and E. coli RNase I) was determinedusing the HUVEC tube formation assay (FIGS. 32 a-h). As is shown inFIGS. 32 a-b while HUVECs incubated in medium in the absence ofangiogenin and RNase (Control, FIG. 32 a), formed only few delicatetubes on the Matrigel surface, in HUVECs incubated in the presence ofangiogenin (Positive Control) massive tubes appeared (FIG. 32 b). Inaddition, while Aspergillus niger RNase B1 and Aspergillus oryzae RNaseT2 did not have any effect on the cells when given alone (NegativeControl, FIGS. 32 c and 32 e, respectively), these RNases clearlyinhibited angiogenin-induced tube formation (FIGS. 32 d and 32 f,respectively). Moreover, RNase T2 was found to have weaker inhibitoryeffect than the RNase B1 (compare FIG. 32 f with FIG. 32 d).Nonetheless, a complete inhibition of tube formation was obtained with10 μM of RNase B1 or 50 μM of RNase T2 (data not shown). On the otherhand, RNase I exhibited a significant inhibitory effect on tubeformation in the absence (Negative Control, FIG. 32 g) or presence ofangiogenin (FIG. 32 h).

Thus, these results clear demonstrate the anti-angiogenic properties ofthe T2 RNases from highly divergent sources. These results are unique tothe instant invention since, up to the present, no publication has shownor suggested the anti-angiogenic activity of RNases of diversephylogenetic origin belonging to the T2 family.

Example 8 RNase B1 Inhibits the Growth of B16F1 and B16F10 MelanomaCells-Induced Tumor

To test the anti-cancer properties of RNase B1, an in vivo melanomamouse model was generated, as follows.

Materials and Experimental Methods

Mice and tumor cells used for the systemic intraperitoneal melanomamouse model—CD BDF1 and Balb/c mice were used for tumor induciton. Mousemelanoma B16F1 (low metastatic) were obtained from INSIGHT toBIOPHARMACEUTICALS LTD. (Rehovot, Israel).

Generation of a melanoma systemic i.p. mouse model—The melanoma mousemodel was generated essentially as described (Geran et al. 1972.Protocol for screening chemical agents and natural products againstanimal tumors and other biological systems. In: Cancer ChemotherapyReports Part 3 Vol 3 No 2). Briefly, 2×10⁶ B16F1 low metastatic cellswere injected into the intraperitoneal (i.p.) cavity of each mouse andthe presence of cancer tumors was evaluated at day 14 following melanomacell injection by an overall view of the mouse and/or histopathologyexaminations.

RNase B1 i.p. administration to the melanoma systemic i.p. mousemodel—Twenty-four hours and 5 days following B16F1 melanoma cellsinjection RNase B1 (5 mg/mouse in 100 μl PBS) or PBS alone was injectedinto the intraperitoneal cavity.

Mice and tumor cells used for the systemic intravenous (i.v.) melanomamouse model—Balb/c mice were used for intravenous administrations ofmouse B16F10 (highly metastatic) melanoma cells.

Generation of a melanoma systemic i.v. mouse model—B16F10 melanoma cells(5×10⁵ or 5×10⁶ cells/mouse) were injected into the lateral tail vein ofBalb/c mice and the presence of cancer tumors was evaluated at day 14following melanoma cell injection by an overall view of the mouse and/orhistopathology examinations.

RNase B1 i.v. administration to the melanoma systemic i.v. mousemodels—RNase B1 (10 mg RNase B1 in 100 μl PBS) or PBS was i.v. injectedinto the lateral tail vein of the melanoma mouse model starting 24 hourafter cells injection and in four days intervals with a total of threeinjections. At the end of experiment, the lungs were removed, weighedand surface metastases were quantified.

Experimental Results

RNase B1 significantly inhibits tumor growth in melanoma systemic i.p.mice models—The effect of RNase B1 in the i.p./i.p. (i.e.,intraperitoneal injection of both melanoma cells and RNase B1) melanomamouse model was scored 14 days post melanoma cell injection byqualitative tumor observations at the abdomen cavity of the treatedmice. As is shown in FIGS. 33 a and c, in both BDF1 and Balb/c mice themelanoma cells induced massive tumors filling the abdominal cavity andspreading over the intestinal gut. On the other hand, in RNaseB1-treated mice only few small tumors were observed (FIG. 33 b, d),demonstrating the tumor growth-inhibitory effect of RNase B1. In thisexperiment the total amount of RNase B1 was 10 mg/mouse. Similar resultswere obtained when mice were treated by a single injection of 10 mgRNase B1 or by 10 daily injections of 1 mg RNase B1 (data not shown).

RNase B1 significantly inhibits metastasis/colonization and growth ofhighly metastatic malignant melanoma tumors in melanoma systemic i.v.mice models—In the i.v./i.v. (i.e., intravenous injection of bothmelanoma cells and RNase B1) melanoma mouse model the melanoma cellsinjected in the tail vein lead to development of lung metastases inabout two weeks. As is shown in FIGS. 34 a-c, two weeks following 5×10⁵B16F10 melanoma cell injection multiple metastases were observed in thePBS-injected mice (FIG. 34 a) as compared with only few metastases inthe RNase-treated mice (FIG. 34 b). A quantitative determination of thenumber of metastases revealed a significant decrease 76% (P<0.001, FIG.34 c) in the RNase B1-treated mice. When the initial bolus of 5×10⁶cells/mouse was used, the metastases in the PBS-injected mice werehighly intense and too dense to be counted (data not shown). In thesemice measurements of the lung weight (FIG. 34 d) and tumor size (FIG. 34e) revealed significant reductions of 25% in lung weight (P<0.01, FIG.34 d) and tumor size (P<0.001, FIG. 34 e) in the RNase B1-treated miceas compared with the PBS-treated mice.

These results therefore convincingly demonstrate that RNase B1 can beused to efficiently inhibit the growth and metastasis of highlymetastatic malignant melanoma tumors.

Example 9 RNase B1 Inhibits Tumor Growth, Lung Metastases and MMP-2Production in A375SM-Injected Melanoma Mouse Models

To further substantiate the capability of RNase B1 to inhibit the growthof cancer tumors, the RNase B1 was injected into A375SM melanomacells—induced mouse models, as follows.

Materials and Experimental Methods

Mice used for generating melanoma mouse model—Male athymic Balb/c nudemice were purchased from the Animal Production Area of the NationalCancer Institute, Frederick Cancer Research Facility (Frederick, Md.).The mice were housed in laminar flow cabinets under specificpathogen-free conditions and used at 7-9 weeks of age.

Melanoma cells—The highly tumorigenic and metastatic human melanomaA375SM cell line was obtained from MD Anderson Cancer Center, Houston,Tex. To prepare tumor cells for inoculation, the cells in exponentialgrowth phase were harvested by brief exposure to a 0.25% trypsin/0.02%ethylenediaminetetraacetic acid solution (w/v). The flask was sharplytapped to dislodge the cells and supplemented medium was added. The cellsuspension was pipetted to produce a single-cell suspension. The cellswere washed and resuspended in Ca²⁺/Mg²⁺-free Hanks' balanced saltsolution (HBSS) to the desired cell concentration. Cell viability wasdetermined by trypan blue exclusion, and only single-cell suspensionsof >90% viability was used.

Generation of melanoma mouse models—Subcutaneous (s.c.) tumors wereproduced by injecting 10⁵ tumor cells/0.1 ml HBSS over the rightscapular region of the mice. Growth of subcutaneous tumors was monitoredby weekly examination of the mice and measurement of tumors withcalipers. The mice were sacrificed 5 weeks following the melanoma cellinjection, and tumors were frozen in OCT compound (Sakura Fineter,Torrance, Calif.), or formalin fixed and then processed forimmunostaining and Hematoxylin and Eosin (H&E) staining.

Generation of experimental lung metastasis—To form lung metastases 10⁶tumor cells in 0.1 ml of HBSS were injected into the lateral tail vein(i.v.) of nude mice. The mice were sacrificed 60 days following melanomacell injection, and the lungs were removed, washed in water, and fixedwith Bouin's solution for 24 hours to facilitate counting of tumornodules. The number of surface tumor nodules was counted under adissecting microscope.

RNase B1 administration—Both the subcutaneous and intravenous melanomato cell injected mice were treated every other day with either 1 mg/100μl of RNase B1 aqueous solution or with phosphate-buffered saline (PBS)by intraperitoneal (i.p.) injection.

CD31 and MMP-2 immunohistochemical analysis—Sections of frozen tissueswere prepared from tumor xenografts. The tissue section slides wererinsed twice with PBS, and endogenous peroxidase was blocked by the useof 3% hydrogen peroxide in PBS for 12 minutes. The samples were thenwashed three times with PBS and incubated for 10 minutes at roomtemperature with a protein-blocking solution consisting of PBS (pH 7.5)supplemented with 5% normal horse serum and 1% normal goat serum. Excessblocking solution was drained and the samples were incubated for 18hours at 4° C. with a 1:100 dilution of monoclonal rat anti-CD31 (1:800)antibody or a 1:200 dilution of anti-MMP-2 (PharMingen, San Diego,Calif.). The samples were then rinsed four times with PBS and incubatedfor 60 minutes at room temperature with the appropriate dilution ofperoxidase-conjugated anti-mouse IgG1, anti-rabbit IgG, or anti-rat IgG.The slides were rinsed with PBS and incubated for 5 minutes withdiaminobenzidine (Research Genetics, Huntsville, Ala.). The sectionswere then washed three times with distilled water and counterstainedwith Gill's hematoxylin (Sigma-Aldrich Co. St Louis, Mo.). For thequantification of microvessel density (MVD), ten fields of the CD31stained samples were counted at 100× magnification. Sections (4 μmthick) of formalin-fixed, paraffin-embedded tumors were also stainedwith H&E for routine histological examination.

Experimental Results

RNase B1 treatment significantly reduced tumor size in A375SM melanomacells-injected mice—The effect of RNase B1 on the tumor growth of humanmelanoma cells was determined in two sets of nude mice. In the first setof nude mice (n=5), A375SM melanoma cells (5×10⁵) were injectedsubcutaneously and three days later, animals injected with tumor cellswere subsequently i.p. injected every other day for 30 days with 1 mg ofRNase B1 or control PBS. Tumor cells in the animals treated with PBSgrew progressively and produced large tumors reaching the size up to 700mm³ mean volume (FIG. 35, Control). In contrast, treatment with to RNaseB1 reduced tumor growth to maximum 100 mm³ mean volume during the sameperiods of time (FIG. 35, RNase B1). In the second set of nude mice(n=8) the mice were injected and treated exactly as described for thefirst set and showed exactly the same effects of RNase B1 on tumorgrowth (data not shown).

RNase B1 treatment significantly reduced the incidence and number oflung metastasis in A375SM melanoma cells-injected mice—To determine theeffect of RNase B1 on metastasis of human melanoma cells, A375SM 10⁶cells were injected intravenously into nude mice to produce experimentallung metastasis. Five days later, animals injected with tumor cells werealso i.p. injected with 1 mg of RNase B1 or control PBS every other dayfor 60 days. It was found that both the incidence and number of lungmetastasis of A375SM cells were reduced in RNase B1-treated mice, whencompared with the control group. In control mice, A375SM cells producednumerous lung metastases (median, 65; range, 16 to 200), whereastreatment with RNase B1 significantly inhibited the ability of A375SMcells to form metastasis in nude mice (median, 10; range, 0 to 75;P<0.05).

RNase B1 treatment decreases the expression of MMP-2 in A375SM melanomamouse models—To determine whether RNase B1 suppresses the expression ofMMP-2 in vivo, tissue sections of tumor xenografts were subjected toimmunohistochemical analysis using an MMP-2 specific antibody. MMP-2staining was observed in control-injected A375SM tumors, but wassignificantly decreased in RNase B1-treated tumors (data not shown).Thus, RNase B1 significantly inhibited the expression of MMP-2 in vivoin melanoma cells.

Altogether, these data demonstrate that treatment of mice with RNase B1leads to suppression of tumor growth and metastasis, and to inhibitionof synthesis of angiogenesis factors such as MMP-2.

Example 10 Local Subcutaneous Administration of RNase B1 InhibitsAngiogenesis

Since MMP-2 is an important angiogenic factor, the present inventorshave determined whether RNase B1 could affect angiogenesis in vivo, asfollows.

Materials and Experimental Methods

Local induction of angiogenesis—Gel foams impregnated with 100 ng/spongeangiogenin were subcutaneously implanted in both sides of a nude mouseand following 2 days the mice were intraperitoneally injected 7 times,every two days, on one side with RNase B1 (250 μM RNase B1 in 100 μl)and on the other side with PBS.

Immunofluorescent staining of CD31/PECAM-1—Frozen gelfoam specimens(obtained from Pharmacia & Upjohn, Peapack, N.J.) were sectioned (10-12μm), mounted on positively charged slides and air-dried for 30 minutes.The sections were then fixed for 5 minutes in cold acetone following bya 5-minutes incubation in a solution of 1:1 acetone:chloroform and anadditional 5-minutes incubation in acetone alone. The samples were thenwashed three times with PBS, incubated for 20 minutes at roomtemperature with a protein-blocking solution containing 4% fish gelatinin PBS, and incubated for 18 hours at 4° C. with a 1:800 dilution of ratmonoclonal anti-mouse CD31 antibody (Pharmingen, San Diego, Calif.).Following antibody incubation the slides were rinsed three times withPBS (3 minute each) and incubated for 1 hour in the dark at roomtemperature with a 1:200 dilution of a secondary goat anti-rat antibodyconjugated to Goat anti-rat Alexa 594 (Molecular Probes Inc., Eugene,Oreg.). Samples were then washed three times with PBS (3 minute each)and then mounted with Vectashield mounting medium for fluorescence withHoechst 33342, trihydrochloride, trihydrate 10 mg/mL solution in water(Molecular Probes Inc., Eugene, Oreg.). Immunoflorescence microscopy wasperformed using a Zeiss Axioplan microscope (Carl Zeiss, New York, N.Y.)equipped with a 100-W HBO mercury bulb and filter sets from Chroma, Inc.(Burlington, Vt.) to individually capture red, green, and bluefluorescent images. Images were captured using a C5810 Hamamatsu colorchilled 3CCD camera (Hamamatsu, Japan) and digitized using Optimasimaging software (Silver Springs, Md.). Images were further processedusing Adobe PhotoShop software (Adobe Systems, Mountain View, Calif.).Endothelial cells were identified by red fluorescence.

TUNEL assay—For terminal deoxynucleotidyl transferase-mediated dUTP-nickend-labeling (TUNEL) assay, a Klenow-FragEl kit (Oncogene Cambridge,Mass.) was used. Sections (10 μm) were deparaffinized in xylene,followed by rehydration in to gradual ethanol solutions (100, 100, 95and 70% ethanol—5 min each). The preparations were subjected to aproteinase K treatment for 15 min, extensively washed in PBS, andpreincubated at room temperature in an equilibration buffer supplied bythe manufacturer. After tapping off excess liquid, terminaldeoxynucleotide transferase and digoxigenin-11-dUTP were applied to thepreparations. The slides were incubated at 37° C. for 1 hour, washed inpre-warmed stop/wash buffer at 37° C. for 30 minutes, and then washedthree times in PBS. Specific staining was achieved by the addition ofantidigoxigenin antibody carrying peroxidase as the reporter conjugate.After counterstaining, the slides were mounted under a glass coverslip,and the brown apoptotic cells were visualized by light microscopy.

Experimental Results

RNase B1 inhibits angiogenin-induced development of blood vessels—Totest the capacity of RNase B1 to inhibit angiogenesis in vivo,angiogenin impregnated gel foams were subcutaneously implanted at bothsides of a nude mouse, followed by i.p. injections of RNase B1 or PBS.As is shown in FIG. 37, while angiogenin-PBS treatment resulted inmassive development of blood vessels (FIG. 37, Angiogenin), theangiogenin-RNase B1 treatment resulted in a significant reduction in theblood vessels (FIG. 37, Angiogenin and RNase B1).

These results demonstrate the ability of RNase B1 to inhibitangiogenesis in vivo.

RNase B1 reduces the tumor MVD in A375SM-induced tumor—Tumor-associatedneovascularization as indicated by microvessel density (MVD) wasdetermined by immunohistochemistry using an anti-CD31 antibody. Asignificant reduction in tumor MVD per field was observed followingtreatment with RNase B1 as compared with control tumors. The mean numberof MVD was 12±5 in RNase B1-treated A375SM tumors as compared with 43±7for control, untreated A375SM-induced tumors (data not shown). Moreover,the number of TUNEL-positive tumor cells was inversely correlated withMVD in the studied tumors. The number of tumor cells undergoingapoptosis was higher in the RNase B1-treated animals than in tumors incontrol mice. Thus, the percentage of apoptotic cells was 31.2±7.3% inRNase B1-treated A375SM melanoma tumors. In contrast, the percentage ofapoptotic cells to was 2.2±1.1% for control A375SM tumors.

Thus, these results demonstrate that RNase B1 treatment significantlydecreased melanoma tumor-associated neovascularization and increasedapoptosis of tumor cells.

Example 11 RNase B1 Reduces Microvessel Density in DMH Model

Materials and Experimental Methods

Rats—Male Wistar rats aged 6 weeks and weighing about 160 g wereobtained from the Charles River-derived outbred male rat.

DMH—1,2-Dimethylhydrazine (DMH) was purchased from Sigma (St. Louis,Mo., USA). DMH was dissolved immediately before use in a solution of1.5% EDTA in PBS and the pH of the solution was brought to pH 6.5.

Generation of DMH-models—DMH was intramuscularly (i.m.) injected to rats(40 mg/kg body weight). At injection time rat weighted 250 gr.

Tumors were induced in rats using dimethylhydrazine (DMH) administeredin subcutaneous injections of DMH (15 mg/100 g body weight, once a weekfor 5 weeks). Control groups received injections of PBS plus the vehicle(EDTA).

Analysis of DMH-models—The rats were first anesthetized with ether andthen sacrificed. From each rat, the colon was excised and the aberrantcrypt foci (ACF) or tumors were counted and measured. Tumors fromcontrol and from RNase B1-treated rats were fixed and embedded inparaffin and the tumor sections were further analyzed by histopathology(H&E staining), immunostaining with anti-CD-31 antibody for blood vesselmonitoring or TUNEL assay for apoptosis.

Immunofluorescent staining of CD31/PECAM-1—was performed as described inExample 10, hereinabove. Briefly, paraffin sections (10-μm) from tumorsgenerated in DMH or DMH+RNase B1-treated mice were subjected to CD31immunostaining using the PECAM-1 (H-300) antibody (sc-8306, Santa CruzBiotechnology, Inc. Santa Cruz, Calif.). Blood vessels in median tumorcross sections were counted and their diameters were measured. In eachtumor, the ratio between blood vessel total area and tumor-section areawas calculated. The experiments were to repeated twice, and eachtreatment was applied to 6-to-10 rats.

Experimental Results

Tumor-associated neovascularization decreased in RNase B1-DMH treatedmice—CD31 immunostaining revealed the presence of blood vessels withinthe tumor and served as a basis for calculation of the microvesseldensity (MVD). As is shown in FIGS. 42 a-b, RNase B1 administration wasfound to significantly reduce the number of blood vessels (angiogenesis)per tumor.

Example 12 RNase B1 Inhibits Invasiveness/Colonization Capacity ofMelenoma, Colon and Mammary Carcinoma Cells In Vitro

Since RNase B1 was shown to affect cancer cell's morphology and actinorganization the present inventors have further examined whether RNaseB1 also affects cell motility, as follows.

Material and Experimental Methods

Colon carcinoma (HT-29) or breast cancer cells (ZR-75-1)invasion/colonization assay—HT-29 colon cancer cells or ZR-75-1 cellswere treated in the presence or absence of 1 and 10 μM RNase B1 for 4days. Wells and Matrigel-coated inserts of a commercially available24-well invasion chamber (Becton Dickinson, Bedford, Mass.) wererehydrated in 0.5 ml of serum free medium overnight and processedaccording to the manufacturer's instructions. Half ml of HT-29 control(i.e., in the absence of RNase B1) or RNase B1-treated cell suspensionscontaining 2.5×10⁴ cells each was added to the top of the chambers and0.750 ml of DMEM media containing 10% FCS were added to the lowerchamber. The invasion chambers were incubated for 22 hours in a 37degrees centrigrade cell culture incubator. The non-invading cells onthe upper surface of the membrane of the insert were removed byscrubbing. The cells on the lower surface of the membrane were stainedwith Diff-Quik™ stain. The membranes were fixed and the cells werecounted at ×200 magnification under a light microscope. The assay wascarried out in triplicate.

Matrigel-coated filter invasion assay—A375SM cells were treated in thepresence or absence of RNase B1 and following 22 hours the number ofcells invaded the Matrigel-coated filter was counted.

MMP-2 release from A375SM cells—Metastatic A375SM cells (5×10³) weregrown in Complete Eagle's minimum essential medium (CMEM), were platedin six-well plates and allowed to attach for 24 hours. Cells weretreated for 4 days with 1 or 10 μM RNase B1, or PBS. Treatment for 4days was found to be optimal for the RNase B1 to affect MMP-2 release.On day 5, CMEM was removed and replaced with serum-free mediumovernight. The supernatant was collected and centrifuged and mediaaliquoted in 500 μl samples and stored at −20° C. Total MMP-2 wasdetermined by the quantikine MMP-2 immunoassay kit (R&D Systems Inc,Minneapolis, Minn.). Samples were diluted 10-fold in a diluent suppliedby the kit. MMP-2 was measured according to the supplier instructionsand the results were adjusted to cell number.

MMP-2 Collagenase activity (Zymograms)—MMP-2 activity was determined onsubstrate-impregnated gels. HUVECs cells or metastatic A375SM cells(5×10³) were plated in six-well plates and allowed to attach for 24hours. Cells were treated for 4 days with 1-5 or 10 μM RNase B1, or PBS.Treatment for 4 days was found to be optimal for the RNase B1 to affectMMP-2 activity. On day 5, CMEM was removed and replaced with serum-freemedium overnight. The supernatant was collected, the volume adjusted forcell number, loaded, and separated on gelatin-impregnated (1 mg/ml;Difco, Detroit, Mich.) sodium dodecyl sulfate/8% polyacrylamide gelsunder nonreducing conditions, followed by 30 minutes of shaking in 2.5%Triton X-100 (BDH, Poole, UK). The gels were then incubated for 16 hoursat 37° C. in 50 mmol/L Tris, 0.2 mol/L NaCl, 5 mmol/L CaCl₂ (w/v) at pH7.6. At the end of the incubation, the gels were stained with 0.5%Coomassie G 250 (Bio-Rad, Richmond, Calif.) in methanol/acetic acid/H₂O(30:10:60). The presence of MMP-2 Collagenase activity is seen as awhite band on blue gels.

Experimental Results

RNase B1 reduces HT-29 or ZR-75-1 invasiveness in vitro—As is shown inTable 6, hereinbelow, HT-29 and ZR-75-1 cells were able to penetrate theMatrigel-coated filters. Surprisingly, RNase B1 was found tosignificantly and dose-dependently (1 and 10 μM) inhibit theinvasiveness/colonization capacity of colon carcinoma cells.

TABLE 6 The effect of RNase B1 on HT-29 or ZR-75-1 invasiveness RNase B1concentration Cell lines Control 1 μM 10 μM HT-29  963.7 ± 95.7 623.3 ±104.2 223.7 ± 13.1 ZR-75-1 1297.7 ± 62.5 784.0 ± 51.5  271.7 ± 16.6Table 6: The Effect of RNase B1 on ZR-75-1 breast cancer and HT-29-coloncancer cell invasiveness through Matrigel-coated filters. In HT-29 andZR-75-1 cells, RNase B1 at 1 μM (P < 0.05 and P < 0.01, respectively)and 10 μM (P < 0.01 and P < 0.001, respectively) inhibited cellinvasiveness in a dose-responsive manner.

RNase B1 reduces A375SM invasiveness in vitro—A375SM cells were treatedin the presence or absence of 1 or 10 μM RNase B1 and the degree of cellinvasiveness was measured in matrigel-coated filters. As is shown inFIG. 38, a significant dose-dependent effect of RNase B1 on theinhibition of A375SM invasiveness was observed. While in untreatedA375SM cells (control) 1216±68 cells penetrated Matrigel-coated filters,725±59 or 211±14 A375SM cells treated with 1 or 10 μM RNase B1penetrated the Matrigel-coated filters.

RNase B1 significantly inhibits the secretion of MMP-2—The level ofMMP-2 release was determined in vitro from A375SM cells. As is shown inFIG. 48, a dose-dependent effect of RNase B1 in inhibiting total MMP-2release was obtained, with the maximal inhibitory effect observed in thepresence of 10 μM RNase B1.

MMP-2 collagenase activity reduces following RNase B1treatment—Zymograms of the supernatant of A375SM or HUVEC cells wereused to determine MMP-2 activity. As is shown in FIG. 49 a-b, while bothuntreated A375SM (FIG. 49 a) and HUVEC (FIG. 49 b) cells exhibit astrong MMP-2 activity, RNase-treated cells exhibited a dose-dependentdecrease in band intensity of the 72 kDa MMP-2, suggesting decreasedMMP-2 activity. Similarly, a dose-dependent decrease was also observedin MMP-9 activity in the RNase B1-treated cells (FIG. 49 b).

Altogether, these results clearly demonstrate theanti-angiogenic/anti-cancer characteristics of RNase B1.

Example 13 Intravenous and Intraperitoneal Administrations of RNase B1Reduce Tumor Size in HT-29 Colon Cancer Models

Materials and Experimental Methods

Generation of an s.c./i.v. colon cancer xenograft model—Tumors of humancolon cancer origin (HT-29) were grafted into 4-5 week old nude mice(CD-1 nu/nu) males, weighing 18-20 grams at onset of experiment. HT-29cells (2×10⁶/mouse) were injected subcutaneously (s.c.) into the mice,at the left hip. T2 RNase (RNase B1, 5 mg in 100 μl PBS) or PBS alonewas injected intravenously (i.v.) into the tail vein 24 hours, 5 days,and 10 days after administration of the colon cancer cells. After 15days, the mice were sacrificed and the tumors or the area of injectionwere assessed by histopathological examination.

Paraffin sections of control and T2 RNase-treated tumors were stainedwith hematoxylin and eosin (H&E) and with Klenow-FragEl kit (Oncogene,Cambridge, Mass.) for evidence of apoptosis.

Generation of an s.c./i.p. colon cancer xenograft model—Colon cancercells (HT-29) and nude mice (CD-1 nu/nu) were as for the s.c./i.v.model. At the 1^(st) day of experiment, cells were injected into theleft hips of mice (10⁶ cells/mouse). RNase B1 was injected into theperitoneal cavity, starting from day 2 and every other day. Twoexperiments were performed. In the first experiment, RNase B1 at dosesof 1 and 5 mg/injection in 100 μl PBS was applied. In the secondexperiment, doses of RNase B1 ranged between 0.01-1 mg/injection wereused. PBS alone was injected as control. The tumors were excised at day30 of experiment, for size measurements and histopathologicalexaminations. Tumor volume was calculated using the equation(length×width)/2.

Experimental Results

Intravenous administration of RNase B1 can efficiently reduce tumorweight—In RNase B1-treated mice, a 60% reduction in tumor weight wasobserved compare to control (FIGS. 36 a-b). These results show that theintravenous administration of RNase B1 is highly effective in inhibitingcolon carcinoma tumor growth.

Intraperitoneal administration of RNase B1 efficiently reduces tumorsize—In the sc/ip mice model, RNase B1 significantly inhibited thegrowth of HT-29 derived-carcinoma (FIGS. 39 a-b). At the therapeuticdoses of 1 and 5 mg/injection (50 and 250 mg/kg), tumor volume wasreduced by 44% and 41% respectively (P<0.05, FIG. 39 b). At 0.001, 0.01,0.1, 0.5 and 1 mg/injection (0.05, 0.5, 5, 25 and 50 mg/kg,respectively), tumor volume was reduced by 3% 41.5%, 34.4%, 51.1% and62.2% respectively (FIG. 39 a), as compared to control (P<0.05). Theseresults are consistent with the previous in vitro experiments with HT-29cells, in which RNase B1 at concentrations ranged between 1-4 μg/100 μl(0.25-1 μM) had similar inhibitory effect on the rate of clonogenicity.Thus, these results demonstrating the preventing (FIG. 39 a) as well asthe therapeutic (FIG. 39 b) effect of RNase B1 on colon tumor size.

RNase B1 accumulates in the peritoneum of the treated mice—As is shownin FIGS. 40 a-c, immunohistochemial staining using rabbit anti-RNase B1and FITC-conjugated goat anti rabbit revealed the presence of RNase B1in the RNase B1-treated mice (FIG. 40 b) but not the PBS treated mice(FIG. 40 c). In addition, RNase B1 immunostaining of the cross sectionsof the treated mice revealed the accumulation of RNase B1 onto the basalmembrane of the tumor blood vessel (FIGS. 41 a-c).

Thus, these results demonstrate that RNase B1 enters the peritoneum andthen finds its way towards the basal membrane of tumor blood vessels.

It is worth mentioning that in the intraperitoneal mode ofadministration appears to be non-toxic to the nude mice, since the bodyweight and other behavioral parameters remained equal to those ofuntreated mice throughout the experiment.

Example 14 RNase B1 Enhances Apoptosis in Cancer Animal Models Materialsand Experimental Methods

TUNEL assay—was performed as described in Example 10, hereinabove.

Melanoma mouse models—B 16F1-induced melanoma mouse models were togenerated as described in Example 8, hereinabove.

Colon cancer mouse models—DMH and HT-29 colon cancer models aredescribed in Examples 11 and 13, hereinabove, respectively.

Experimental Results

RNase B1 enhances apoptosis in B16F1-induced melanoma cell tumors—Todetermine the rate of apoptosis within the tumors, tumor tissue sectionsfrom RNase B1-treated or untreated melanoma mouse models were subjectedto a TUNEL assay. As is shown in FIGS. 43 a-b, in the RNase B1-treatedmice the apoptosis rate was significantly increased as compared with theuntreated melanoma mouse model.

Enhanced apoptosis rate in RNase-treated DMH-colon cancer rat model—Asis shown in FIG. 44 a, tumors obtained from the DMH colon cancer modelexhibited negligible levels of apoptosis. On the other hand, tumorsobtained from RNase B1-treated rats exhibited a significant level ofapoptosis (FIG. 44 b). When 10 different microscopic fields at ×200magnification were counted in three different tissue sections the numberof apoptotic cells was found to be 2.01±0.2/microscopic field incontrol, untreated tumors and 37±5/microscopic field in tumors of theRNase B1-treated animals.

High proportion of apoptotic cells in HT-29 colon carcinoma-induced micewhich were treated with RNase B1—As is shown in FIGS. 45 a-d, whileuntreated HT-29-induced colon carcinoma mice exhibited vital andactively dividing nuclei (FIGS. 45 a and c), the RNase-treated micedisplayed condensed cytoplasm and nuclei (FIG. 45 b) and high proportionof apoptotic cells (FIG. 45 d).

Altogether, these results demonstrate the significant apoptosis effectof RNase B1 on the various cancer-induced animal models, and suggest theuse of RNase B1 in treatment of disorders of apoptosis and abnormalaccumulation of cells, such as cancer and inflammatory/ischaemicdisease.

Example 15 RNase B1 and Taxol Exhibit A Synergistic Effect on InhibitingRelative Tumor Volume in Colon Cancer Xenograft Models

To test whether RNase B1 can inhibit tumor growth on established tumorsand to determine whether RNase B1 displays an additive or synergisticeffect with Taxol, a common colon cancer cytotoxic drugs, RNase B1 wasemployed together with Taxol on colon cancer xenograft models, asfollows.

Materials and Experimental Methods

The following protocol is based on Fujii T., et al. Anticancer Res.23:2405-2412 (2003).

Animals—Nude male mice (balb/c nu/nu), 7 week old, weigh 22 gram each.

Cancer cells—Human colon cancer LS174T. LS174T cancer cells (1×10⁶ cellsper mouse per 100 μl medium) were subcutaneously injected into the nudemice.

RNase B1 administration—started when tumors are palpable (10-13 daysafter cell injection), and consisted of 15 i.p. injections every otherday. RNase B1 doses were 10 and 1000 micrograms/100 μl/injection (0.5and 50 mg/kg, respectively).

Taxol preparation and administration—Fifty mg Taxol were dissolved in 18ml propylene glycol and 3 ml ethanol, following which 9 ml of water wereadded. Taxol was administered by i.p. injections for 5 consecutive daysout of 7 days over a period of 3 weeks, starting when tumors arepalpable (13 days after cell injection).

Treatments—The combined treatment was applied as followed:

Control: PBS or propylene glycol+ethanol−3 mice for each vehicle.

RNase B1: 50 mg/kg RNase B1 (1000 μg/injection).

RNase B1+Taxol: 50 mg/kg RNase B1 and 5 mg/kg Taxol.

Taxol: 5 mg/kg TAXOL (100 μg/injection).

During the experiment, the tumors were measured twice a week using acaliper. Tumor volume was calculated using the equation(length×width²)/2. Each mouse was tagged and monitored individually.Relative tumor volume (RTV) was defined as RTV=V_(i)/V₀, where V_(i) wastumor volume at any given time and V₀ was that at the time of initialtreatment. At the end of experiments, samples were taken to histology.

Experimental Results

The highly angiogenic LS174T cell line was used in the combined RNase B1and Taxol treatment. As is clearly shown in FIG. 46, while Taxondisplays only marginal effect on the relative tumor volume (RTV), RNaseB1 exhibits a significant effect in inhibiting tumor growth. However,when RNase B1 and Taxol were concomitantly injected into the mice, asignificant inhibition of tumor growth was observed, demonstrating asynergistic, rather that additive effect of RNase B1 to taxol treatment.Moreover, the RTV at endpoint were 158.2, 195.3, 61.4 and 13.43 incontrol, Taxol, RNase B1 and RNase B1+Taxol treatments, respectively(FIG. 46), demonstrating, for the first time, an efficient suppressionof tumor growth.

These results indicate that combined treatment has a greater potentialfor inhibiting tumor growth rate than each drug alone. These resultsfurther demonstrate the ability of RNase B1 to inhibit the growth ofpre-established tumors.

Example 16 Actin-Binding in Diverse Rnase B1

The actin-binding activity of diverse RNase of the T2 family wasinvestigated.

Materials and Experimental Methods

Actin-Western Blot—This assay is based on the method described by Hu etal. 1993 (Actin is a binding protein for angiogenin. Proc Natl Acad SciUSA. 90:1217-21). Briefly, RNase B1, Angiogenin, RNase I (1 μg of eachprotein) and actin (as a positive control) were subjected to SDS-PAGE,followed by Western Blot analysis. The nitrocellulose membrane wasblocked overnight with BSA and incubated for another overnight in 5 mlbuffer containing 25 μg G-actin, following which a monoclonal mouseanti-actin IgM was applied followed by HRP-conjugated goat anti mouseIgM Actin (Ab-1) Kit, CAT #CP01-1EA (Oncogene)]. The HRP-derived signalswere detected by the Super-Signal® enhanced-chemiluminescence system(ECL, Pierce).

Experimental Results

Actin-binding properties of bacterial and A. niger T2 RNase—As is shownin FIGS. 47 a-b, the actin-Western Blot analysis revealed a strongassociation of RNase B1 to G-actin, demonstrating its actin-bindingcapacity.

Example 17 Cellular Localization of Rnase B1 Materials and ExperimentalMethods

Immunocytochemistry staining of cultured Cells—A375SM or HUVEC cellswere cultured on chamber slides and fixed with cold acetone for 20minutes. The slides were rinsed and then blocked with 4% gel fish for 20minutes at room temperature. The chamber slides were incubated withprimary antibody overnight at 4° C. After being washed with 0.01 M pH7.4 PBS three times, the cells were incubated for 60 minutes withFITC-conjugated antirabbit secondary antibody diluted with 4% gel fishin PBS. For HUVEC CD31 immunostaining, the specimens were incubated for18 hour at 4° C. with a 1:800 dilution of rat monoclonal anti-mouse CD31antibody (Pharmingen, San Diego, Calif.). After the samples were rinsedwith PBS three times for 3 minutes each, the slides were incubated for 1hour in the dark at room temperature with 1:200 dilution of secondarygoat anti-rat antibody conjugated to Goat anti-rat Alexa 594 (MolecularProbes Inc., Eugene, Oreg.). Samples were washed three times with PBS, 3minutes each, and mounted with Vectashield mounting medium forfluorescence with Hoechst 33342, trihydrochloride, trihydrate 10 mg/mLsolution in water (Molecular Probes Inc., Eugene, O). The slides wereviewed on Zeiss laser scanning confocal microscope. Z-sections andXZ-sections were obtained from 3D scanning by using LSM510 software.

Experimental Results

RNase B1 is localized within the HUVEC cells—As is shown in FIGS. 50a-s, RNase B1 gradually penetrates the cell membrane which is detectedby the red label of CD31 immunostaining and enters into the cell.

RNase B1 reaches the nucleus of A375SM melanoma cells—As is shown inFIGS. 51 a-c, while within two hours of exposure to RNase B1 the cellmorphology changes and becomes round (FIG. 51 a), after four hours theRNase B1 is seen within the cytoplasm of some cells (FIG. 51 b) andfollowing eight hours reaches the cell nuclei (FIG. 51 c). It is worthmentioning that many of the melanoma A375SM cells exhibit apoptoticcharacteristics following 8 hours of exposure to RNase B1 (FIG. 51 c).

Thus, these results demonstrate that RNase B1 enters both HUVEC andmelanoma A375SM cells and induces apoptosis in the melanoma cells. Theresults to also clearly demonstrate that RNase B1 penetration into thecells is a slow process lasting several hours, in contrast to theimmediate response seen in pollen tubes.

Example 18 Cloning the Gene Encoding Rnase B1

The mechanism by which RNase B1 is produced by the fungus Aspergillusniger is not known yet. The genes coding Aspergillus oryzae RNase T₂(Ozeki et al. 1991. Cloning and nucleotide sequence of the genomicribonuclease T2 gene (mtB) from Aspergillus oryzae. Curr Genet.19:367-73) and Rhizopus niveus RNase Rh (Horiuchi et al. 1988. Primarystructure of a base non-specific ribonuclease from Rhizopus niveus JBiochem 103:408-18), which are RNase T2-family members, have beencloned. Understanding the genetics of RNase B1 is of great importance,since the potential for recombinant protein production would beaugmented. To study the amino acid sequence of RNase B1, it was firstdigested with trypsin and chymotrypsin and fragments were analyzed byLiquid Chromatography Mass Spectra (LC-MS) and compared againstdatabase. Some peptide sequences were found to be 100% homologous to A.saitoi RNase M (Accession No: P19791; Watanabe et al. 1990. Primarystructure of a base non-specific and adenylic acid preferential fromAspergillus saitoi. J. Biochem. 10:303-310) and together they comprised60% of the protein sequence (data not shown). It is interesting tomention that the taxons saitoi and phoenicus were previously classifiedas variants of the A. niger group (Al-Musallam, A. 1980. Revision of theblack Aspergillus species. PhD. Thesis, State University, Utrecht,Netherlands). The identified amino acid sequences enabled the design ofDNA oligonucleotide primers which can be used on genomic DNA. Since mostamino acids are encoded by at least two nucleotide codons, the presentinventors used degenerate primers having low degeneracy, according to A.niger codon usage, as follows: Forward primer-5′-TTYTGGGARCAYGARTGGAAY-3′ (for amino acids F107-N112) (SEQ ID NO:1)and reverse primer -5′-CCYTTIACRTTRAARTARTARTA-3′ (reverse complementfor amino acids Y200-K206) (SEQ ID NO:2). The letter “Y” refers to anyof the nucleotides C or T and the letter “R” refers to any of thenucleotides A or G. Deoxyinosine (I) replaces any of the fournucleotides.

Experimental Results

The major 400-bp band obtained after PCR amplification (FIG. 52) wasexcised from the gel and cloned into pGEM-T vector (Promega) for DNAsequence analysis. The 300 nucleotides that were obtained (SEQ ID NO: 4)created an open reading frame for 100 amino acids (SEQ ID NO: 5, FIG.53), which matched the F107-K206 of RNase M almost completely (exceptposition 123 where glutamic acid replaced aspartic acid in RNASE B1 andRNase M, respectively). This sequence makes the mid region of the gene.Further experiments are currently being conducted to complete the fullgene sequence of RNase B1 from Aspergillus niger B1 (CMI CC 234626).

Example 19 Cloning and Purification of the Human RNase 6PL Protein

Deletion of a region of chromosome 6 in humans (6q27) has beenconsidered to be associated with several human malignancies (Cooke etal. 1996. Genes Chromosomes Cancer 15:223-233; Saito et al. 1992. CancerRes 52:5815-5817). It was found that this region contains the putativetumor suppressor RNase6PL gene which shares homology with the RNase T2family (Trubia et al. 1997. Genomics 42:342-344; Acquati et al. 2001.Meth Mol Biol 160:87-101), including RNase B1. Due to the anti-cancercapacities of T2-family RNases, human RNase T2 represents a highlyadvantageous agent for treating cancer by virtue of its being ofendogenous human origin, and thereby being optimally non-immunogenic andnon-toxic when administered to humans. To test the possibility that thehuman RNAse 6PL protein can be used as an anti-cancer agent, the humanRNAse 6PL protein was synthesized, as follows.

Materials and Experimental Methods

Expression of human RNAse6PL protein in Pichia pastoris—The sequence ofthe gene for the RNase6PL was identified in the human genome project(genomic sequence: GenBank Accession No. NT_(—)007422; cDNA sequence:GenBank Accession No. NM_(—)003730). Out of 28,751 bp of the full gene,only 719 bp form the open reading frame. To produce a recombinantRNAse6PL protein the synthetic 719 bp cDNA (GenArt GmbH, Germany) wasligated into the pPIC9K plasmid and was to further transformed into thePichia pastoris yeast. The recombinant yeasts were grown under inductiveconditions (i.e., in the presence of 0.5% methanol), and the obtainedcolonies were tested for the presence of the RNAse6PL insert.

Purification of the human RNase6PL protein from the yeast clone—Topurify the recombinant RNase6PL protein a positive colony (i.e., acolony containing the RNAse6PL insert cDNA) was fermented according tomanufacturer's instructions (Invitrogen Inc.) and the medium supernatantwas heat treated (10 minutes at 90° C.) followed by buffer exchange bydialysis using 20 mM Tris-HCl pH 7. The relatively pure protein wasloaded on a Q Sepharose column in a Fast Protein Liquid Chromatography(FPLC) (Amersham Pharmacia Biotech, Buckinghamshire, U.K.), using a 5-mlcolumn at a flow rate of 5 ml/min. To elute the active RNase6PL protein,an NaCl gradient from 0 to 1 M was applied to the column (for a timeperiod of 20 minutes) and eluted proteins were collected in 0.5 mlfractions.

Experimental Results

The structure of the human RNase 6PL gene—The sequence of the gene forRNase6PL was identified in the human genome project. Out of 28,751 bp ofthe full gene, only 719 bp (arranged in 9 exons) form the open readingframe.

Identification of an RNAse6PL yeast clone—The coding region of RNase6PLwas cloned into the pPIC9K plasmid of the Pichia pastoris yeast and onecolony was found to contain a gene insert of about 750 bp, demonstratingthe cloning of a recombinant RNase6PL in yeast.

Recombinant RNase6PL is produced in the Pichia pastoris yeast—Thepositive colony was cultured under adequate conditions to over-expressthe recombinant RNase6PL. The yeast protein extract was passed through aQ Sepharose column in a Fast Protein Liquid Chromatography (FPLC)(Amersham Pharmacia Biotech, Buckinghamshire, U.K.). The eluted proteinwas tested on an SDS-PAGE for the presence of an RNase6PL protein. As isshown in FIG. 54, a 27-kDa protein was obtained from the elutedfraction, consisting of 15 mg of purified RNase6PL protein.

The purified recombinant RNase6PL protein is thermostable at varyingtemperatures—The thermostability of the recombinant protein wasevaluated on an SDS-PAGE following the incubation for 10 minutes of theRNase6PL at increasing to temperatures from 55-100° C. The recombinantRNase6PL was stable at all temperatures tested.

Recombinant human RNase6PL is catalytically active at varioustemperatures—The catalytic activity of the recombinant RNase6PL wastested in vitro by its ability to degrade RNA. The recombinant proteinwas capable of degrading RNA following the incubation of the protein(for 10 minutes) at varying temperatures from 55-100° C.

Altogether, these results demonstrate the simple and efficientpurification of recombinant RNase6PL protein by denaturation by hightemperature and subsequent isolation and purification on a column. Inaddition, the thermostability and activity at increasing temperaturedemonstrate that the recombinant protein exhibits the characteristicproperties typical to members of the RNase T2 family.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by an accession number mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

What is claimed is:
 1. A method of preventing, inhibiting and/orreversing proliferation, colonization, differentiation and/ordevelopment of cancer cells in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a ribonuclease of the T2 family wherein the ribonuclease T2binds actin in either its active or non-active ribonucleolytic form. 2.The method of claim 1, wherein said proliferation, colonization,differentiation and/or development of said cancer cells is atransformation of a benign tumor to a malignant tumor.
 3. The method ofclaim 1, wherein said preventing, inhibiting and/or reversingproliferation, colonization, differentiation and/or development of saidcancer cells is preventing or inhibiting metastatic spread of tumorcells.
 4. The method of claim 1, wherein said proliferation,colonization, differentiation and/or development of said cancer cells istumor angiogenesis.
 5. The method of claim 1, wherein said ribonucleaseof the T2 family comprises a T2 ribonuclease protein having a molecularweight of between 24-36 kDa.
 6. The method of claim 1, wherein saidribonuclease of the T2 family is devoid of ribonuclease activity.
 7. Themethod of claim 1, wherein an actin binding activity of saidribonuclease protein is boiling stable.
 8. The method of claim 1,wherein administering to the subject said therapeutically effectiveamount of said ribonuclease of the T2 family is effected by parenteraladministration.
 9. The method of claim 1, wherein said cancer cells areor are associated with a disease selected from the group consisting ofblastoglioma, Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer,prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neckcancer, bladder cancer, breast cancer, colorectal cancer, thyroidcancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma,leukemia, lymphoma, Hodgkin's disease and Burkitt's disease.
 10. Themethod of claim 1, wherein administering is by an administration modeselected from the group consisting of oral administration, topicaladministration, transmucosal administration, parenteral administration,rectal administration and by inhalation.
 11. The method of claim 1,wherein said ribonuclease of the T2 family is A. niger B1 RNase.
 12. Themethod of claim 1, wherein said ribonuclease of the T2 family isselected from the group consisting of RNase T2, RNase Rh, RNase M, RNaseTrv, RNase Irp, RNase Le2, RNase Phyb, RNase LE, RNase MC, RNase CL1,RNase Bsp1, RNase RCL2, RNase Dm, RNase Oy and RNase Tp, RNase HI0526,RNase I, Rnase Irp1, RNS2, RNase 3, RNase 1, RNase LX, RNase 6PL. 13.The method of claim 1, wherein said ribonuclease is a T2 ribonucleasefrom an organism selected from the group consisting of Aeromonashydrophila, Haemophilus influenzae, Escherichia coli, Aspergillusoryzae, Aspergillus phoenicis, Rhisopus niveus, Trichoderma viride,Lentinula edodes, Irpex lacteus; Physarum polycephlum, Arabidopsisthaliana, Lycopersicon esculentum, Nicotiana alata, Malus domestica,Pyrus pyrifolia, Momordica charantia, Gallus gallus, Rana catesbeiana,Drosophyla melanogaster, Crassostera gigus, Todarodes pasificus and Homosapiens.